Physiological and Morphological Basis for Differences in Growth, Water Use and Drought Resistance Among Cercis L. Taxa Dissertat

PHYSIOLOGICAL AND MORPHOLOGICAL BASIS FOR DIFFERENCES IN GROWTH, WATER USE AND DROUGHT RESISTANCE AMONG CERCIS L. TAXA

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Petra Sternberg, M.S.

Graduate Program in Horticulture and Crop Science

The Ohio State University

2012

Dissertation Committee:

Daniel K. Struve, Advisor

David J. Barker

James D. Metzger

Daniel A. Herms

Copyrighted by

Petra Sternberg

2012

Abstract

Water is one of the principle factors determining the structure and species composition of the vegetation occupying a given site. Plantings in constructed sites are particularly problematic as urban environments magnify stresses that are common to most landscapes.

Often, trees are selected predominantly for their aesthetic contribution to the landscape, with little consideration of their adaptation to the site. Selecting trees with improved drought resistance and reduced water use may be the best strategy to improve survival, growth and health of trees in urban and suburban landscapes. However, the variety of plant characteristics that contribute to drought resistance and the variation in site characteristics makes plant selection for dry sites difficult. Cercis are small to medium sized and often multi-stemmed trees. Cercis canadensis L. is a valuable commercially- produced landscape tree in the United States. The aim of this study was to investigate the inter-taxa variation in drought resistance and water use within the genus Cercis, thereby providing information as to their usefulness for plantings in urban landscapes and to facilitate breeding for improved water use and drought resistance.

The genus Cercis currently includes ten recognized species native to the warm, north- temperate zones of North America and Eurasia. The exact number of species and their delimitation remains controversial.

The first objective was to describe the Cercis taxa to be used in subsequent experiments

(Chapters 3 and 4), to compare them to Cercis described in the literature and describe morphological differences that can influence drought resistance. Growth habit and leaf characteristics were measured and principal component analysis employed to describe variation in the observed characteristics. The taxa described were representative of their species and/or variety. Cercis taxa differed widely in their growth habit and leaf characteristics and showed adaption to their native environment. Cercis employed common as well as taxa-specific strategies to deal with similar environmental conditions.

The persistence of the leaf characteristics under non-limiting soil moisture conditions indicated that the characteristics typical of the taxa grown in their native environment were under genetic control. The observation that some characteristics on the Eastern

North American taxa espressed great variability indicated introgression of these charateristics and that the taxa are still in the process of speciation.

The root system needs to enable plants to meet their transpirational demand and is a key component of plant adaption to xeric environments. Thus, the second objective was to evaluate water use characteristics and morphology of seven Cercis L. taxa of different origins and to determine the growth strategies of the taxa in terms above versus below ground biomass under non-limiting soil moisture conditions. Seven Cercis L. taxa were grown from seed in the greenhouse, and 60 days after germination, water use plant-1 day-1 was determined gravimetrically, three times over 72 h. Height, caliper, number of nodes on the main shoot axis and leaf area were measured. Roots were scanned and analyzed with WinRhizo software to determine root length, root surface area and root diameter.

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Dry weight of leaves, shoots and roots was obtained. A principal component analysis showed great differences in shoot and root morphology as well as water use characteristics among taxa. It showed a trend towards decreasing water use per unit plant mass with increasing plant size. No consistent pattern of characteristics employed by mesic- or xeric-adapted taxa could be found. Each taxon uses a unique set of characteristics to adapt to its original environment.

Plants have developed morphological, anatomical and physiological adaptations to resist drought. The third objective was to determine morphological and physiological differences among Cercis taxa in response to water deficit. Seedlings of seven Cercis taxa were submitted to a drought and recovery cycle under greenhouse conditions. Net photosynthesis, stomatal conductance, intrinsic water use efficiency and chlorophyll fluorescence, as well as plant growth and water use were measured. Paraheliotropism was observed in all taxa at varying degrees. All taxa reduced net photosynthesis and stomatal conductance shortly after exposure to limited water availability. Cercis occidentalis and

C. siliquastrum presented with high initial values for net photosynthesis and stomatal conductance and showed a relatively lower decline in both values than other taxa.

Chlorophyll fluorescence declined in some taxa during the drought period, while other taxa where unaffected. Affected taxa showed inclining values for net photosynthesis, stomatal conductance and chlorophyll fluorescence during the recovery period. Taxa in this study are drought-resistant and employ several common drought resistant mechanisms. One is the ability of plants to withstand drought stress without altering the function of the PSII system and/or the capacity of plants to recover from damage another is paraheliotropism. Overall, the Cercis taxa displayed great inter-taxa variation. Some

common drought resistance mechanisms were found, however, other mechanisms, especially differences in growth habit and morphology appeared to be more specific adaptations to the native environment of each taxon; for instance a shrub-like growth habit, consistent with capturing precipitation via stem flow, and/or glaucousness of the leaves. Cercis canadensis var. texensis would be an excellent candidate for inducing drought resistance into a breeding program based on the combination of relatively high drought resistance displayed in these experiments, its upright growth habit and rapid growth rate.

Dedication

I dedicate this dissertation to my loving husband Kurt Bresko

and my adorable son Christopher (“Muckel”).

Thank you for your smiles!

Acknowledgments

I wish to thank everybody whose assistance, advice and support have made the completion of my graduate studies and this dissertation possible.

First, I would like to express my sincere gratitude to my doctoral advisor Dr. Daniel

Struve for his guidance and advice. I would also like to thank my graduate committee,

Dr. David Barker, Dr. James Metzger and Dr. Daniel Herms, for their direction, dedication and invaluable advice along this project.

I am indebted to my parents for their love and emotional support. I am especially thankful to my son, Christopher Bresko, for being the ultimate reason for finishing this dissertation, and to my husband Kurt Bresko, for his love and encouragement. Without this invaluable team the completion of my dissertation would have not been possible.

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Vita

2003 ...... Diplom-Ingenieur Gartenbau (FH), University of Applied Science Osnabrück, Germany

2007 ...... M.S. Horticulture and Crop Sciences, The Ohio State University

2007 to present ...... Graduate Research and Teaching Associate, Department of Horticulture and Crop Science, The Ohio State University

Publications

Sternberg, P. and D.K. Struve, 2008 Cyclanilide foliar applications induce grater lateral branching than pruning in container grown whips. Journal of Environmental Horticulture 26(1): 45-50

Sternberg, P. and D.K. Struve, 2007 Cyclanilide spray increases branching in containerized whip production. Journal of Environmental Horticulture 25(4): 221-228

Struve, D.K., Sternberg, P. , Drunasky, N., Bresko, K.L. and R. Gonzales, 2006 Growth and water use characteristics of six Eastern North American Oak (Quercus) species and the implications for urban forestry. Arboriculture and Urban Forestry 32: 202-213

Fields of Study

Major Field: Horticulture and Crop Science

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Table of Contents

Abstract ...... ii

Dedication ...... vi

Acknowledgments ...... vii

Vita ...... viii

List of Tables...... xii

List of Figures ...... xiv

CHAPTER 1

INTRODUCTION ...... 1

1.1 Statement of the problem and significance ...... 2

1.2 Description and taxonomy of the genus Cercis L...... 4

1.3 Drought stress ...... 12

1.4 Drought resistance mechanisms ...... 18

1.5 Cercis response to drought ...... 31

1.6 Overall objective/hypothesis...... 34

CHAPTER 2

SPECIES DESCRIPTION ...... 36

2.1 Abstract ...... 37

2.2 Introduction ...... 38

2.3 Material and methods ...... 40

2.4 Results ...... 45

2.5 Discussion ...... 58

CHAPTER 3

WATER USE AND MORPHOLOGY OF EIGHT CERCIS L. TAXA - INSIGHTS

INTO SPECIATION ...... 75

3.1 Abstract ...... 76

3.2 Introduction ...... 77

3.3 Material and methods ...... 81

3.4 Results ...... 83

3.5 Discussion ...... 91

CHAPTER 4

PHOTOSYNTHESIS, CHLOROPHYLL FLUORESCENCE AND WATER USE OF

SEVEN CERCIS L. TAXA DURING DROUGHT STRESS AND RECOVERY ...... 94

4.1 Abstract ...... 95

4.2 Introduction ...... 97

4.3 Material and methods ...... 100

4.4 Results ...... 106

4.5 Discussion ...... 142

CHAPTER 5

DISCUSSION ...... 149

BIBLIOGRAPHY ...... 160

List of Tables

Table

2.1 Rotated factor pattern for principal component analysis of growth characteristics of seven Cercis L. taxa...... 46

2.2 Caliper, height and shoot growth of seven Cercis L. taxa...... 49

2.3 Branching habit of seven Cercis L. taxa...... 50

2.4 Rotated factor pattern for principal component analysis of leaf characteristics of seven Cercis L. taxa...... 52

2.5 Lamina characteristics of nine Cercis L. taxa...... 56

2.6 Leaf sinus, vein, petiole and pubescence characters for nine Cercis L. taxa...... 57

3.1 Rotated factor pattern for principal component analysis of plant growth and water use characteristics of seven Cercis L. taxa...... 84

3.2 Plant growth characteristics of seven Cercis taxa L. of different origin...... 86

3.3 Root growth characteristics and shoot-to-root relations of seven Cercis taxa of different origin...... 87

3.4 Water use characteristics of seven Cercis of different origin...... 88

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4.1 Differences in water use in g between watered control plants and non-watered plants of seven Cercis L. taxa during a water deficit and recovery cycle ...... 110

4.2 Plant morphological data of seven Cercis L. taxa at start of a water deficit and recovery cycle...... 116

4.3 Dry weight of seven Cercis taxa at beginning of a water deficit and recovery cycle...... 117

4.4 Changes in caliper size and number of leafless basal nodes for Cercis plants from seven taxa exposed to an 8 d water deficit period and 18 d recovery period. .. 119

4.5 Changes in leaf and shoot dry weight of seven Cercis taxa exposed to a water deficit and recovery cycle...... 124

4.6 Changes in root and total dry weight of seven Cercis taxa exposed to a water deficit and recovery cycle...... 126

4.7 Net assimilation rate (NAR) and relative growth rate (RGR) of seven Cercis taxa exposed to a water deficit and recovery cycle...... 129

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List of Figures

Figure

1.1 Worldwide distribution of the genus Cercis (Coskun 2003, changed) ...... 11

2.1 Description of measurements taken on mature leaves of Cercis L...... 44

2.2 Plotted values of the first three principal components of the growth habit description of Cercis L. taxa...... 48

2.3 Plotted values for the first three principal components of the leaf description of Cercis L. taxa...... 55

2.4 A-G. Photographs of one-year old plants of Cercis L. taxa...... 66

2.5 H-O. Photographs of (a) adaxial and (b) abaxial leaf surface of Cercis L. taxa...... 69

3.1 Plotted values of the first three principal components of plant growth and water use characteristics of Cercis L. taxa...... 85

4.1 Substrate moisture release curve for SunGrow MetroMix and substrate moisture content of non-watered plants and watered control plants of Cercis after 9 d of withholding water ...... 106

-1 4.2 A-G. Intrinsic water use efficiency (μmol CO2 mol H2O) and water use (g) of seven Cercis taxa exposed to a drought and recovery cycle...... 111

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4.3 H-N. Photosynthesis, stomatal conductance and chlorophyll fluorescence of seven Cercis taxa exposed to a drought and recovery cycle...... 133

4.4 Morphological signs of drought stress of seven Cercis taxa...... 139

Chapter 1

INTRODUCTION

1.1 Statement of the problem and significance

Water is one of the principle factors determining the structure and species composition of the vegetation occupying a given site. Its deficit limits plant distribution and productivity at a global scale (Whitlow et al. 1992). Of the environmental stresses encountered by plants in urban and suburban landscapes, drought stress is the one most frequent and most injurious due to its effect on virtually all physiological processes (Kramer 1987, Cregg

2004). Drought stress can be defined as any period during which plant and/or soil water deficit affects the growth and development of plants (Quizenberry 1982). Urban environments magnify stresses that are common to most landscapes and most often, trees are selected for their aesthetic contribution to the landscape, with little consideration of the adaptation to the site. Unlike natural plantings, trees in urban environments are often physically isolated from each other. Street trees face curtailed water supply and excessive demand which prevail in the urban environment (Whitlow et al. 1992). There is along list of conditions occurring creating an extreme microclimate never encountered in nature

(Whitlow and Bassuk 1988). Soil compaction, high soil pH, de-icing salts, waterlogging, lack of water, air pollution, and high summer temperatures can be potentially lethal, either singly or in combination (Whitlow and Bassuk 1988, Whitlow et al. 1992, Ware

1994). Restricted rooting volume and reduced infiltration into soils that are compacted and covered with imperious pavements decreases water supply to the roots. On the other hand are access to ground water and subsurface drainage often eliminated. Atmospheric demand is increased due to heightened vapor pressure deficits due to higher temperatures and lower humidities (Whitlow et al. 1992). Irrigation can mitigate the impact of drought stress but with water restrictions becoming more common throughout the US, increasing

water prices, limited availability and ground water pollution, it is becoming less of an option (Schuch and Burger 1987, Knox 1989). Selecting trees with improved drought tolerance and reduced water use may be the best strategy to improve survival, growth and health of trees in urban and suburban landscapes (Cregg 2004).

1.2 Description and taxonomy of the genus Cercis L.

The genus Cercis belongs to the subfamily Caesalpinioideae of the large plant family

Fabaceae (Leguminosae – legume family). The genus Cercis currently includes ten recognized species scattered widely across the warm, north-temperate zones of North

America and Eurasia (Fritsch et al. 2009, Fig. 1.1). There is some uncertainty as to the exact number of species, and their delimitation remains controversial. The species C. siliquastrum is native to Europe, and five species (C. chinensis, C. chingii, C. chuniana,

C. glabra and C. racemosa) are indigenous to China (Woodward and Williams 1987, Pu

Zou et al. 2008). Cercis griffithii is native to Afghanistan (Raulston 1968). The taxonomy of the Cercis species native to North America is confusing and has undergone several changes in concept. In the traditional concept (i.e. Hopkins 1942) the North American component of the genus Cercis comprised of C. canadensis and C. occidentalis. Three subspecies/varieties of C. canadensis are recognized: C. canadenis var. canadensis throughout the east of the United States, C. canadensis var. texensis, found in east-central

Texas and adjacent Oklahoma, and the Mexican redbud (C. canadensis var. mexicana) found in Trans-Pecos Texas and northeastern Mexico (Hopkins 1942, Isley 1975, Correll and Johnston 1979, Turner et al. 2003).

Cercis L. (Redbud) are trees or shrubs of small to medium size with alternate, undivided, heart- to kidney-shaped, toothless leaves with five to nine conspicuous veins radiating from their base (Everett 1981). Widely cultivated as ornamentals, especially in North

America, their reddish to pink or white flowers open before the growth of the leaves and offer a unique view in early spring due to their development on stem or trunk (Coskun

2003). Isely (1975) offered two alternatives concerning the recognition of Cercis species in the United States: There are either two species, C. canadensis, typically found in more mesophytic habitats and C. occidentalis, typical from more xerophytic habitats or there is just one species with regional varieties. A study by Salatino (2000), involving flavonoids, supports the first alternative, because of the existence of two chemical groups among the

North American species of Cercis, where C. canadensis belongs to the group of kaempferol bearer and C. occidentalis belongs to another group which is devoid of kaempferol. Hopkins (1942) also identified these two species, with C. occidentalis being restricted to areas west of the Rocky Mountains, chiefly to California, but also locally in

Arizona, Utah and Nevada, and C. canadensis with several subspecies and varieties and a much broader range across the eastern and central United States.

The most important Cercis species in the United States is the eastern redbud (Cercis canadensis). It is a valuable landscape tree and produced commercially as a nursery crop

(Nardini et al. 2003). These small to medium sized trees can reach up to 12 m height and are often multi-stemmed. It is described as the hardiest and most-tree like of the redbud species (Raulston 1968). Its leaves are pointed at the tips, of light green color and 7.5 to

15 cm wide. Due to its small size, it is especially suitable for residential gardens (Clark and Bachtell 1992) and other areas where space is limited. It is also used for urban landscapes in parking lots, buffer strips and as street tree. The usage of Cercis for this purpose is limited since the lifetime of Cercis is 15-20 years in urban settings. However, if the tree is not subjected to stresses such as drought, poor soil or stem injury it can be an attractive tree for many decades. Cercis canadensis is native throughout the United States

and one of the most widely distributed redbuds, ranging as far west as southeastern

Nebraska, western Oklahoma and Texas, and southward into east-central Mexico and west-central Florida (Donselman and Flint 1982, Clark and Bachtell 1992). In its cooler northern natural range, C. canadensis is found in areas with high light, mostly open woodlands and along forest edges (Nardini et al. 2003). In the warmer part of its distribution, such as the Arbuckle Mountains, OK, it is found under more shaded conditions (Hopkins 1942, Nardini et al. 2003). According to Clark and Bachtell (1992) the plants occurs on almost any site that is not excessively dry or wet, or has strongly acidic soils. Associated with the wide range of climatic and geographic conditions, C. canadensis shows clinal variation and substantial differences that can be found in dormancy, morphology and hardiness (Donselman 1976, Donselman and Flint 1982,

Banner and Stein 2008).

Traditionally three varieties have been recognized in the United States: C. canadensis var. canadensis which is widespread in the Eastern United States and C. can. var. texensis (S. Watson) M. Hopkins and C. canadensis var. mexicana (Rose) M. Hopkins which are native to more xeric south western environments.

Cercis canadensis var. texensis, the Texas redbud, is found in east-central Texas and adjacent Oklahoma mostly on Paleozoic limestone as xeric pastures, outcrops and hills

(Hopkins 1942, Fritsch et al. 2009). It is considered highly drought resistant and grows well on most soils as long as they are well-drained. It is more tolerant of acidic soils than other redbuds and grows in full or partial sun. Cercis canadensis var. texensis is a small tree, usually 4.5 to 6 m tall (even up to 12 m) with a rounded or vase shape. Kruessmann

(1984) lists its hardiness as USDA zone 8, the USDA considers it hardy in zones 5 to 9A

(Kruessmann 1984, Gilman and Watson 1993). Hopkins (1942) describes the plant as very similar to C. canadensis when growing in the field. Only the leaves differ by being relatively thick, leathery and glaucous, with a typical sheen while leaves of C. canadensis are merely dull green.

Cercis canadensis var. mexicana, the Mexican redbud native range includes Trans-

Pecos Texas and northeastern Mexico where the multi-stemmed tree grows on limestone soils (Fritsch et al. 2009). There is some confusion concerning its botanical name as well as its relationship to other redbuds (Kruessmann 1984). Hopkins (1942) describes this plant as differing from C. canadensis var. canadenis and C. canadensis var. texensis only

“in having young branches and petioles covered with a brownish, very tomentose pubescence”, and considers it an environmentally induced morphology and recommends it should be listed under C. canadensis var. texensis. However, often Mexican redbud is distinguished from the Texas redbud (Raulston 1968, Tipton and White 1995, Banner and

Stein 2008) because is it visibly different from C. canadensis var. texensis, with much smaller leaves and an undulate leaf margin. The plants are finer in texture and densely branched (Raulston 1968). However, the foliage is extremely variable, with either a thin/dull cuticle or a thick/glossy cuticle (Tipton and White 1995). The plant can grow into a 1.5 m shrub or a 3.5 m high tree (Raulston 1990, Ladybird Johnson Wildflower

Center 2010). In its native range, C. canadensis var. mexicana grows on calcareous, limestone based soils (Ladybird Johnson Wildflower Center 2010). It is typically found in xeric environments and is adapted well to dry south-west conditions (Robertson 1976,

Bennett 1987). Hardy in USDA hardiness zone (6) 7, C. canadensis var. mexicana could be an excellent miniature Cercis for small residential areas and garden (Kruessmann

1984, Raulston 1990).

Cercis occidentalis, the Western redbud, is native to California, Arizona, Utah (Davies et al. 2002, National Resources Conservation Center 2010), Nevada (Hopkins 1942) and

Oregon (Raulston 1990). It is considered drought tolerant and can be found in foothill woodlands, chaparral and exposed dry washes as well as in protected valleys (Ballenger

1992, Davies et al. 2002). C. occidentalis is considered drought-tolerant and is adapted to

Mediterranean-like summers (Raulston 1990, National Resources Conservation Center

2010). Although it grows on a variety of soils, it is usually found on well-drained, but coarse and nutrient-poor soils with high pH. It is mostly found growing singly, but in sheltered situations it can occur in shrubby clumps (National Resources Conservation

Center 2010). The plants usually grows as a spreading, multi-stemmed shrub with a dense crown that almost reaches the ground (Brickell and Cathey 2004, National Resources

Conservation Center 2010), but can reach tree size and form in some locations (Raulston

1990). Plants with heights of 1.2 to 1.5 m as well as 3 to 5.5 are included in the natural variation of this species.

In Europe Cercis siliquastrum, the Mediterranean redbud, is commercially important in its natural range throughout the Mediterranean region and southern Europe, from France to Turkey and Afghanistan (Robertson 1976, Rechinger 1986, Davies et al. 2002) but is so widely naturalized that it is difficult to determine its original distribution. Robertson

(1976) speculates it might be native from Turkey eastward to Afghanistan while Raulston

(1990) considers it native from Spain across Southern Europe to Israel. The plant occurs in maquis (shrubland biome in the Mediterranean region) in the Mediterranean region as well as in montane forests in west central Asia (Rechinger 1986, Davies et al. 2002). The tree is also native to Lebanon, where it can be found in the Thermomediterranean zone

(0-500 m altitude) and up to 800 m altitude, then associated with pine and oak forests.

Cercis siliquastrum is well adapted to a semi-arid climate. Where the tree is native, most of the annual rainfall (700-1000 mm) occurs between November and March, only 5% between May and September. C. siliquastrum grows well on a variety of soil textures but does best in soils with a pH above 7.5. It tends to be tolerant of nutrient deficient sites and grows best in full sun. It can withstand hot dry summers as long as soil moisture is adequate in winter and spring (Anonymous 1999). The plant itself, a spreading, sometimes multi-stemmed tree grows up to 12 m in cultivation; however, most plants do not exceed 4.5 to 6 m height (Raulston 1990). Cercis siliquastrum was cultivated by early

Middle Eastern civilizations, and accurately described before 1600 (Raulston 1990). The species is very variable and there seems to be a great ecotypic variation in nature as well as in cultivation and many of these variants have been recognized as either different species or varieties (Raulston 1990, Robertson 1976).

Cercis chinensis, the Chinese redbud, is native and widely distributed in temperate

China. Its natural habitat is limited in eastern China to the hills along the western borders of the coastal provinces Kiangsu and Checkiang. In the south it can be found in Fukien, northern Kwangsi, Kweichow, and northern Yunnan, with 24° N being its approximate southern limit. The hilly district of eastern Szechuan, where the species occurs most

abundantly may be considered the center of development of C. chinensis. It is a temperate zone tree that occurs in hilly districts but is also found in the open country and the margins of woodlands, generally along slopes up to 1200m altitude (Hiu-Lin 1944).

The species was in cultivation before 1850 and is considered the tallest redbud, when grown in the wild with up to 15 m in height and a trunk caliper of 90 to 120 cm. In cultivation, however, C. chinensis is almost always a small, multi-stemmed shrub or tree,

3 to 4.5 m in height. (Raulston 1968, Robertson 1976, Everett 1981). The plants grow strictly upright and have glossy, dark green leaves. In the United States C. chinensis is propagated by microcuttings and somewhat commercially important.

Cercis griffithii, the Afghan redbud, is native to Afghanistan (Raulston 1968) and described to be very similar to and considered by some an ecotype of C. siliquastrum

(Raulston 1990).

C. siliquastrum C. griffithii C. chinensis C. occidentalis

C. canadensis

C. canadensis var. texensis

C. canadensis

1 var. mexicana

Fig. 1.1 Worldwide distribution of the genus Cercis L. (Coskun 2003, changed)

1.3 Drought stress

Plants are frequently exposed to a variety of stresses. Stress can be defined as any environmental factor that may adversely affect growth, development, or productivity

(Levitt 1972, Lichtenthaler 1996). Larcher (2003) defined drought in meteorological terms as a period without appreciable precipitation during which the soil water content is reduced to an extent that plants suffer from water deficit. Lichtenthaler (1996) extended

Larcher’s stress concept of plants by differentiating between eu-stress and dis-stress.

While eu-stress is an activating, stimulating stress and positive for plant development, dis-stress is a severe and real stress that causes damage and therefore negatively affects the plant and its development. Unlike other stresses, drought stress does not occur suddenly, but rather slowly and its intensity increases with time (Munne-Bosch and

Alegre 2004). Drought stress is a multidimensional stress, affecting nearly every plant process, directly or indirectly and has a dramatic impact on physiological processes such as photosynthesis, respiration, protein synthesis and carbohydrate metabolism (Pallardy

2008). Plant growth is very sensitive to water deficits and is reduced by even mild stress

(Kozlowski et al. 1991, Jones 1992). The reduction in growth is mainly caused by a reduction in cell enlargement, which is extremely sensitive to water deficit (Hsiao 1973,

Kramer 1983, Taiz and Zeiger 2002), whereas cell division, although affected by drought stress, is less sensitive (Jones 1992).

Plants have developed many anatomical, morphological and physiological characteristics for resisting drought stress (Flint 1985). Drought resistance is the ability of a plant to withstand periods of dryness, it includes both the avoidance of plant water deficits

(dehydration avoidance) and the tolerance of plant water deficits (dehydration tolerance)

(Larcher 2003). True dehydration avoidance is a mechanism by which plants “escape” the drought by completing their life cycle during a period of high rain fall and soil water availability (Pallardy 1981, Ludlow 1989, Jones 1992, Kozlowski and Pallardy 1997,

Taiz and Zeiger 2002, Larcher 2003) and then survive unfavorable dry conditions in a dehydration tolerant form – such as seeds (Hao et al. 2001). Woody plant species cannot escape from drought, but are physiologically active during times of likely drought and therefore must possess a certain degree of drought resistance. Many taxa have developed several mechanisms allowing them to avoid dehydration by maintaining high plant water status or cellular hydration under drought conditions (Pita et al. 2005, Blum 2005). Since plant functions are relatively unexposed to tissue dehydration, the plant avoids stress.

Dehydration avoidance includes strategies to maximize water uptake, such as deep growing roots or decreasing water loss by both elastic (e.g. stomatal control, leaf movements) and plastic responses (e.g. decreasing leaf size) (Davies 1988, Hao et al.

2001). Plant species avoiding dehydration can therefore be a water “saver” or water

“spender” (Ludlow 1989, Hao et al. 2001). Water “savers” tend to minimize water loss and maintain high leaf water potentials (Ludlow 1989). Water “spenders” on the other hand are very efficient in water uptake and maintain a high transpiration rate, photosynthesis and growth. Plants using the avoidance strategy have good short-term survival in areas where drought periods do not exceed 3-4 months. Their long-term survival, however, is generally poor since the strategy eventually fails to prevent the dehydration of relatively desiccation sensitive tissue, resulting in the death of the plant.

For longer survival under drought plants may show desiccation tolerance responses.

Dehydration tolerant plants are able to maintain their metabolism even at low leaf water potential. Dehydration tolerance seems more important for plant survival than high growth rates (Turner 1986). It should be noted that these strategies are not mutually exclusive and that each species possesses its own strategy to resist drought, which may be closer to an extreme avoidance or extreme tolerance (Ludlow 1989). In general, plants with high dehydration tolerance have poorly developed dehydration avoidance responses.

Generally, dehydration tolerant plants are lower plants such as mosses and bryophytes

(Pallardy 1981). Drought resistant higher plants and most woody plants are desiccation avoiding rather than desiccation tolerant (Levitt 1972).

1.3.1 Adaptation versus acclimation

Kramer (1983) defined adaptation as: “heritable modifications in structure and processes that increase the probability of organisms surviving in a given environment”. Adaptation is the result of natural selection and represents a response to environmental conditions at the population level and is therefore heritable. However, many modifications in the evolution of plants did not originate in order to improve survival but are the result of random mutation and recombination events (Kramer 1983). Steward and Hanson (1980) suggested that these modifications might be beneficial, injurious, or only incidental.

Beneficial modifications that enabled plants to better survive and reproduce in a given environment were preserved by natural selection. Some characteristics without apparent benefit may also survive, simply because they have never been subjected to negative selection. Plants subjected to frequent drought stress often show characteristics such as an extensive root system or a thick cuticle. These characteristics however, could have

occurred randomly in populations in both wet and dry habitats, but were only preserved in dry habitats because of their added benefit for the plant. This does not mean however, that adaptations necessary for survival in a changing environment occurred and thus many species became extinct because they were not able to adapt (Kramer 1983).

The difference between adaptation and acclimation is sometimes hard to distinguish.

Many characteristics of plants adapted to drought are constitutive rather than stress- induced and determine plant shape and development such as the extent of the root system or xylem properties (Blum 1984, Passioura 2002, Trimble 2007). Some however, respond to drought in some degree. Wax and hair development, cuticle thickness as well as leaf reflectivity have been reported to increase with increasing drought (Jones 1992) showing acclimation which is defined as “phenotypic modification produced by variation in environmental factors” (Kramer 1983). Acclimation is a response at the individual level; it is purely phenotypic and reversible. The ability to acclimate however is an adaptation

(Kramer 1983). Examples of drought acclimation include reduced leaf area, osmotic adjustment and an increase in shoot-to-root ratio. Drought hardening is a process of acclimation and is frequently used by growers of herbaceous and woody plants to

“harden” seedlings before transplanting in the field to increase their survival rate (Kramer

1983). Plants that have been exposed to previous drought stress suffer less injury from subsequent drought events than plants not previously exposed. Besides watering plants less frequently, drought stress can be produced by root pruning or exposing the plants to full sun.

How long a plant can withstand a drought without damage depends on the plant, the water holding capacity in its root zone, as well as the atmospheric conditions that affect 15

the rates of evaporation and transpiration (Kramer 1983). Often the relationship between annual precipitation and annual evaporation is used to determine the humid or arid nature of an area. This, however, can only be an indication, since at the plant-level it only matters that there is water available when they need it, which is mostly during the growing season (Larcher 2003).

Lichtenthaler (1996) differentiates between short-term and long-term stress effects as well as between low stress events, which can be partially compensated for by acclimation, adaptation and repair mechanisms, and strong stress or chronic stress events which can cause considerable damage and may eventually lead to cell and plant death. In nature, plants are faced with either a slowly developing drought (within days to weeks or month) or a short-term water deficit (hours to days). In the case of a slowly developing drought, plants can shorten their life cycle and “escape” dehydration or acclimate by optimizing their resource gain long term. If the drought develops rapidly, plants can minimize their water loss or exhibit metabolic protection against the damaging dehydration effects. Therefore, fast and slow developing desiccations trigger different physiological responses of adaptation (McDonald and Davies 1996). But the importance of time in how a plant reacts to drought also depends dramatically on the environment and the genotype of the plant (Chaves et al. 2003). If, for example, rainfall is significant but erratic in summer, plants that have a large leaf area, or are able to develop a large leaf area quickly, can take advantage of these occasionally wet summers. One acclimation strategy in these conditions is the capacity for both vegetative growth and extensive flowering over a long period of time. If on the other hand, precipitation only occurs

during winter and spring, only plants that can complete their life cycle quickly will be able to produce seed for the next generation (Taiz and Zeiger 2002).

1.4 Drought resistance mechanisms

1.4.1 Desiccation avoidance

Desiccation avoidance mechanisms promote water homeostasis by either increasing water absorption to replace water lost by transpiration or by restricting water loss from the plant (Sharp and Davies 1989). Many of these adaptations of woody plants have been identified in leaves, stems and roots.

MORPHOLOGICAL EFFECTS

Relative capacity for soil water absorption

Roots are the main apparatus for meeting the transpirational demands of a plant. A deep, wide-spread and much branched root system maximizes the uptake of available water from the soil, allowing the plant to avoid drought injury for a longer period of time between precipitation events (Kramer 1983, Graves 1996). Some tree species develop extensive root systems as seedlings before shoot growth is favored, regardless of the soil moisture status; in others the allocation of photosynthetic energy preferentially to the root system is a response to drought (Graves 1996). However, drought stress generally inhibits root growth. Hardly any evidence exists that total root mass increases with drought. The shoot-root ratio is known to decrease under drought conditions but these changes are mainly due to a reduction in shoot growth, not an increase in root growth (Graves 1996,

Blum 2005). However, even if total root mass is reduced in a drying soil, root length and depth may still be increased (Blum 2005). Therefore, shoot-root ratio or total root dry matter may not provide the information needed to make selections for drought resistant plants. 18

Plant resistance to water transport

Water movement within plants is largely governed by a gradient in water potential

(Pallardy 2008). Leaf transpiration creates a negative pressure in the leaf end of the hydraulic system. This negative pressure propagates down the system to the roots and leads to negative pressure within the conduits (Wei et al. 1999a, Wei et al. 1999b). The gradient in water potential that is needed to sustain a constant flow of water in the plant increases with resistance to water flow in roots, stem and leaves (Pallardy 1981). Xylem structure and function vary widely among taxonomic groups (Pallardy 2008). This suggests that the liquid flow resistance can have a major impact on the maximum transpiration rate and the drop in water potential from the soil to the leaves. Especially in tall trees, the xylem resistance and its resulting physiological effects can be significant

(Pallardy 1981). Species with a wide natural range may be genetically adapted to specific environments by specialized alteration in their xylem structure.

The plant’s hydraulic system can be separated into a root and a shoot component. The root component generally accounts for 20 to 90% of the total resistance (Tsuda and Tyree

1997 and references within). Differences in the root proportion, their anatomy and the depth at which they grow are largely responsible for the variability (Steudle 2002). The higher hydraulic conductance, the higher stomatal conductance that can be maintained without risking dehydration or xylem embolism and therefore, the higher the photosynthetic capacity (Lo Gullo and Salleo 1988). However, drought decreases water availability and reduces root-to-soil contact (Lo Gullo et al. 1998), imposing restriction on water uptake. This increased mismatch of water uptake/ transport, and transpiration leads to an increased tension of the xylem sap, therefore increasing the risk of air entering 19

the conduits and triggering xylem cavitations and finally embolism (Tyree and Sperry

1989). These air embolisms in the conducting xylem were found to decrease water conductivity (Tyree and Sperry 1988, 1989). The extent of embolism of the conducting conduits varies with species, season and different parts of the same tree. The vulnerability to embolism under tension even seems to be correlated with a species distribution across habitats of varying water availability (Tyree and Evers 1991, Sperry and Sullivan 1992).

Control of transpiration

Shoot growth and leaf size and morphology

When grown in a common, low stress environment, plants originating from seeds collected from the more xeric habitats showed slower growth rates than those collected from the mesic areas of the natural range of the species (Pallardy 1981). Slower shoot growth and less leaf area, reduce the danger of dehydration due to drought. In general, leaves of desert and Mediterranean angiosperm species have smaller leaves than temperate zone angiosperm trees, and leaves produced during a drought are smaller than those produced under adequate soil moisture (Hinckley et al. 1981). Pallardy (1981) noted that within a temperate-zone angiosperm species, smaller leaves were associated with more xeric seed sources. Small leaves have a thin boundary layer and therefore a low boundary layer resistance. This reduces the difference between leaf and air temperature by facilitating sensible heat exchange at the expense of increased transpiration rates due to decreased boundary layer resistance.

Leaf cuticle

The leaf cuticle has several effects on the water balance of plants. It primarily functions as a hydrophobic barrier restricting water loss, but also alters the spectral properties as well as the energy balance of the leaf by its physical structure (Quariani et al. 2000). The covering of hydrophobic substances over the plant cuticle that can impart a dull-white or bluish-grey cast is referred to as glaucousness. Its morphological and chemical composition varies widely and depends not only on the plant taxon but also on its geographical location, the environmental conditions and the stage of development

(Tomaszenwski 2004). It may increase the efficiency of stomatal control by reducing cuticular water loss of the plant (Quariani et al. 2000). The water loss through the leaf epidermis (i.e. loss through the cuticle plus loss due to incomplete stomatal closure) in water-stressed plants may comprise up to 50% of total transpiration during the day and

100% at night. It is important to note that it is the cuticle thickness and rather than cuticular wax content that determines the resistance of the cuticle to water loss

(Schönherr 1976). Glaucousness increases reflection of incoming radiation at the UV and

400-700 nm wavelength to the extent that transpiration and leaf temperature are reduced without a reduction in stomatal conductance (Blum 2005). During severe water deficits, plant survival depends on the ability to restrict such water losses (Quariani et al. 2000).

Genetic differences in cuticular effectiveness have also been identified, suggesting that selection and breeding could improve cuticular effectiveness (Nagarajah 1979). However, while these reflective properties are beneficial under drought conditions, they are also often associated with a reduced photosynthesis and yield potential (Sanchez et al. 2001) and ultimately slow growth rate.

Leaf senescence / abscission

Drought-induced leaf senescence contributes to nutrient remobilization during stress, thus allowing the rest of the plant (i.e. the youngest leaves, fruits or flowers) to benefit from nutrients accumulated during the life span of the leaf. In addition, drought-induced leaf senescence, especially when accompanied by leaf abscission, avoids large losses through transpiration, thus contributing to the maintenance of a favorable water balance of the whole plant (Munne-Bosch and Alegre 2004). During drought, deciduous plants drop their leaves rather than investing energy in protection of foliage from excessive dehydration. These plants then re-foliate when normal water levels resume (Griffin

2002). Although the leaf abscission response can provide protection against lethal desiccation to vital meristems, it occurs with the cost of lost photosynthetic potential

(Pallary 2008).

Stomatal control and anatomy

The quantity and pattern of water loss from a plant can be affected by stomatal adaptations such as a reduction in number and/or size of stomata, stomata that are repressed into crypts and even alterations in the stomatal response to internal or environmental water stress (Pallardy 1981).

Species variation in the number of genotypes has been identified (Nunes 1967,

Ceulemans et al. 1978). These variations involve the stomatal response to either leaf water stress or indirectly to soil water stress. Kozlowski (1981) and Pallardy (1981) suggest that although the data reporting genetic variation in stomatal response to soil moisture and atmospheric factors is fragmentary, that if plant adaptations for drought

resistance can be correlated with their field performance under drought, a ground work for breeding of woody plants for drought resistance will have been established.

The reduction of water loss by stomatal closure is one effective dehydration avoidance mechanisms (Kramer 1983). Especially in xerophytes, where most responsive stomata are associated with low cuticular transpiration. Many xerophytes have high transpiration rates while the soil is moist, but as the soil dries, the stomata eventually remain closed all day. Partial stomatal closure decreases transpiration and increases the leaves water potential as leaves become more hydrated (Tardieu and Davies 1993). This allows the plant leaves to maintain their water status within a narrow range. The closure of stomata is one of the earliest responses to drought stress. This mechanism protects the plants from extensive water loss, which can lead to cell dehydration. Stomata often close before there are any changes detectable in leaf water potential (Gollan et al. 1985, Socias et al. 1997) because stomatal closure is more related to soil moisture status than leaf water status.

Therefore the root system plays a vital role in this mechanism, since it is the only plant structure that can be directly affected by soil moisture supply (Taiz and Zeiger 2002). It is established that abscisic acid (ABA) is synthesized in the roots as a response to soil drying (Davies and Zhang 1991) and travels as a root-to-leaf signal through the transpiration stream and induces stomatal closure in the leaves (Medrano et al. 2002).

However, the role of ABA is not simple and stomatal conductance is not only controlled by soil moisture but by other internal and external factors. A direct correlation between the ABA content in the xylem and stomatal conductance has only been seen in some cases (Correia et al. 1995, Socias et al. 1997). Stomatal closure increases progressively as the drought progresses and is followed by a parallel decrease of net photosynthesis 23

(Medrano 2002). Stomatal adjustment seems to be especially beneficial in dehydration tolerant species native to arid to semiarid regions, where survival is more important than growth (Ludlow 1980).

However, stomatal closure at high leaf water potentials cannot necessarily be identified as a useful adaptation in all species, simply because any beneficial effect on plant water relations coincides with the cost of reduced net photosynthesis as leaf temperature increases due to stomatal closure (Pallardy 2008).

PHYSIOLOGICAL EFFECTS

Photosynthesis

One major effect of drought is reduced photosynthesis. This can be caused by a decreased efficiency of the carbon fixation process, but also by a decline in leaf expansion and premature leaf senescence (Dubey 1997, Pallardy 2008). The reduction in photosynthesis may be due to the stomatal limitation (stomatal closure and the increased resistance to diffusion of CO2) or non-stomatal limitation (inhibition of the photosynthetic apparatus)

(Yordanov et al. 2000, Pallardy 2008). Stomatal closure is the first line of defense and most important factor in controlling carbon fixation (Yordanov et al. 2000). It is much quicker than other mechanisms against dehydration like changes in root growth, leaf area, chloroplast ultrastructure, and pigment-proteins. It was thought that only in cases of severe stress is photosynthesis more limited by the capacity of chloroplast for fixation of

CO2 than by the increased diffusive resistance (Herppich and Peckmann 1997). However,

some evidence suggests that photophosphorylation (Havaux et al. 1987), RuBP regeneration (Gimenez et al. 1992, Gunasekera and Berkowitz 1993) and Rubisco activity (Medrano et al. 1997) are impaired under drought and Tezera et al. (1999) showed that even under mild drought impaired photophosphorylation and ATP synthesis were the main factors limiting photosynthesis in sunflower. Lawlor and Cornic (2002) presented two models of the photosynthetic response of plants to water deficit. If an inhibition of photosynthesis by reduced relative water content can initially be overcome by elevated CO2 levels, plants are displaying a Type 1 response. For a Type 1 response, reduced relative water content (RWC) does not affect the photosynthetic metabolic capacity per se, but it is the stomatal closure that constitutes the primary limitation. With further declining RWC, mesophyll-level inhibition is more likely since photosynthesis cannot be completely restored to pre-stress levels, regardless of the CO2 concentration. In contrast, Type 2 response plants show increasing levels of mesophyll limitation as RWC declines. In Type 2 plants, maximal photosynthesis cannot be restored by elevated CO2 levels even under mild drought stress. The relative importance of stomatal and non- stomatal inhibition of photosynthesis during drought varies with the drought tolerance of tree species. Kubiske and Abrams (1993) found that drought increased stomatal limitations of photosynthesis in both xeric and mesic species, but that in wet-mesic species the mesophyll limitation was at least important in limiting photosynthesis as was stomatal limitation. A general trend of decreased relative mesophyll limitation was observed in xeric species, suggesting that drought tolerant species have a capacity for high carbon fixation. Therefore, Lawlor and Cornin (2002) suggest that the more drought tolerant species belong to the Type 1 response plants.

As discussed earlier, drought resistance associated characteristics seem to be more important for plant survival than high growth rates (Turner 1986). However, the maintenance of a high gas exchange capacity under drought conditions may favor both survival as well as productivity according to Ni and Pallardy (1991). Krueger and van

Rensburg (1995) suggested the survival and recovery following a drought is directly related to the plant’s ability to maintain positive photosynthesis during a slowly developing drought. The recovery will depend on species, drought severity and duration as well as relative humidity (Pallardy 2008). A significant amount of time is required for leaf cells to fully repair the photosynthetic machinery.

1.4.2 Desiccation tolerance

Osmotic adjustment

The accumulation of solutes by the cell has long been proposed as an adaptive mechanism for drought tolerance (Serraj and Sinclair 2002). A variety of common solutes, such as sugars, organic acids, amino acids, and inorganic ions (mostly K+) are responsible for the decrease in osmotic water potential (Taiz and Zeiger 2002). In most plants this decrease is limited to about 0.2 to 0.8 MPa. The decrease in cell osmotic potential helps maintain water absorption, contributing to sustaining physiological processes such as stomatal opening, photosynthesis and expansion growth at water potentials that would otherwise be inhibitory (Kramer 1983, Pallardy 2008). Only a decrease in osmotic potential that is greater than can be caused by an increase in solute potential due to dehydration is considered osmotic adjustment (Kramer 1983). It occurs during a slowly developing drought, but has also been seen as a response to diurnal 26

midday water stress. It is not detectable in all plants or in all cultivars within a species but has been observed in many woody plants (Kramer 1983, Pallardy 2008). Hinckley et al.

(1981) concluded that osmotic adjustment in leaves is only of minor importance as an adaptation to drought in several species of shrubs. Some species (Acer saccharum

(Bahari et al. 1985) and C. canadensis (Buxton et al. 1985) do not show any osmotic adjustment.

Cell wall elasticity

Turgor can also be regulated by changes in cell wall elasticity (Kozlowski and Pallardy

2002). As water is lost from plants, inelastic tissues lose turgor faster than elastic tissues.

Under water stress conditions an increase in tissue elasticity may be desirable under conditions where turgor maintenance is adaptive (e.g. for growth). Decreased elasticity under drought, on the other hand, maintains higher levels of tissue relative water content as water potential declines, protecting tissues from low relative water content and possibly toxic concentrations of ions. Therefore, both increases and decreases in cell wall elasticity may be advantages, depending on the particular environment of the plant

(Pallardy 2008).

Efficiency mechanisms

Water use efficiency

Water use efficiency (WUE) is often erroneously equated with drought resistance and the improvement of crop yield under stress (Blum 2005). At the whole plant level, water-use efficiency is defined as the ratio of dry matter produced per unit evapotranspiration

(Turner 1997). Depending on the objective of the experiment certain vegetative plant parts, such as roots, might be neglected in the calculation, leading to lower water use efficiencies when compared to calculations based on the entire plant (Kramer 1983). Also of importance is whether only water lost by transpiration is used or the combined losses of transpiration and evaporation (evapotranspiration). When dry matter production is only related to the amount of water lost by transpiration it is called transpiration efficiency

(Pita et al. 2005). The use of evapotranspiration is more realistic with respect to agriculture, but also results in higher variability since evaporation is affected by the degree of leaf shading of the soil and the frequency of soil wetting.

At the leaf level, instantaneous water-use efficiency is defined as ratio of photosynthetic carbon gain to transpiration. The two measures of WUE are not equivalent and depending on the timescale involved in measurements, the WUE definition may vary among plant physiologists. In the study of woody plant gas exchange, it is often defined as “instantaneous”. The daily value of WUE would be different because of the nightly consumption of photosynthate by respiration and because the measurements of gas exchange may not have been made under growing conditions representative of the whole plant. It is therefore important to define WUE and interpret it cautiously (Pallardy 2008).

Water use efficiency is an important indicator for a plant’s adaptation to water limitations

(Larcher 2003). However, high water use efficiency does not necessarily equal drought resistance (Blum 2005). Strong relationships appear to exist between WUE and the mode of photosynthesis for CAM (crassulacean acid metabolism), C3 and C4 species. C4 plants tend to exhibit higher WUE than C3 species, especially under water-stressed, hot, high- light conditions. CAM species show greater WUE when operating in CAM mode than when operating in C3 mode. Stomatal closure is often considered the first line of defense against drought. During the early stages of drought, stomatal closure may increase WUE

(Taiz and Zeiger 2002) as the proportional decrease in net photosynthetic rate is less than the decline in stomatal conductance, leading to an increase in intrinsic WUE. However,

WUE usually decreases as drought stress becomes more severe due to the inhibition of photosynthesis in the dehydrated mesophyll cells (Ni and Pallardy 1991, Larcher 2003).

A higher WUE may be related to either higher growth and photosynthesis rates or to lower growth rates due to stomatal closure (Pita et al. 2005). Unfortunately, selecting for improved WUE may often be the same as selecting for low productivity, as instantaneous

WUE increases as stomata close and net photosynthesis decreases. A substantial difference in WUE among species also seems to exist that is independent of the mode of photosynthesis. Differences in instantaneous WUE among several C3 conifer and hardwood species were observed by DeLucia and Heckathorn (1989) and Ni and Pallardy

(1991). Their data did not support the claim that high WUE is a general adaptation of drought-tolerant plants. High water use efficiency is mostly a function of reduced water use rather than a net improvement in plant production or biochemistry of assimilation.

Therefore, it may constitute a marker for reduced water use commonly achieved by

reduced growth and leaf area, but should not be used indiscriminately for the selection of plants with improved resistance to drought stress or improved yield under stress conditions (Blum 2005). High WUE may only be of benefit if the conserved soil water is available for later uptake (DeLucia and Heckathorn 1989). However, if the conserved water is taken up by competing species or is ultimately lost by evaporation, high WUE might even be a disadvantage (Ni and Pallardy 1991). In xeric habitats with mixed natural communities the preemption of available water by a species may be at least as important as high WUE to survival and productivity (Chabot and Bunce 1979). However, the selection of plants with high WUE may be advised when water availability limits plant productivity and economic considerations prohibit intensive cultural operations, such as irrigation (Ni and Pallary 1991). Of course, superior WUE must be accompanied by the ability to survive a drought as well as maintain a growth rate during drought that is acceptable (Pallardy 2008). Often, breeders of crop plants sought plants with a high yield under drought conditions. But this is too broad of an objective for a breeding program

(Kramer 1983). Instead, physiological and morphological characteristics that are likely to improve yield under various conditions need to be identified.

1.5 Cercis response to drought

1.5.1 North American taxa

Davies et al. (2002) proposed that eastern redbud and Mexican redbud are related through a common xerophytic ancestor, and that the mesophytic eastern redbud is a reversion of a xerophytic form. The eastern redbud is typically found in the understory of temperate mesophytic forest. However, it could have retained some xerophytic characteristics that provide competitive advantages in xeric environments like dry woodland understory, ridge top and rock outcrop habitats where it is also can be found. This would explain why the eastern redbud proved to be surprisingly heat and drought tolerant in a study by

Griffin (2002) and responded similarly to the more xerophytic Mexican redbud. Griffin

(2002) studied the effect of heat and drought on photosynthesis and water relations of C. canadensis var. canadensis and C. canadensis var. mexicana. Both ecotypes proved to be extremely drought resistant. The rate of net photosynthesis decreased at a similar rate in both ecotypes but Mexican redbud maintained generally greater net photosynthetic rates than eastern redbud. Mexican redbud also maintained greater water use efficiency under increasing drought than C. canadensis var. canadensis. It had a high rate of net photosynthesis and did not immediately reduce stomatal conductance during drought stress. It was also observed that the Mexican redbud tended to shed leaves under severe drought conditions. The frequently reported solute accumulation in drought stressed plants did not occur. Osmotic potential at full turgor was unaffected by drought in both redbud ecotypes.

Tipton and White (1995) reported that the leaves of dull- as well as glossy-leaved

Mexican redbud (C. canadensis var. mexicana) exhibited several xeromorphic characters

when compared to the eastern redbud (C. canadensis): smaller, thicker leaves with thicker cuticles, more cuticular wax, greater hydrated water content on a leaf area basis and a higher specific leaf mass. Dull-leaved Mexican redbuds differed from glossy- leaved ones only in a thicker adaxial cuticle lacking crystalline wax on the surface. A higher water loss rate was found for detached leaves of eastern redbud compared to

Mexican rebud, but only on a dry weight basis, not on a leaf area basis. This suggested that Mexican redbud did not adapt to xeric conditions by reducing water loss through cuticular transpiration but reflected the benefits of smaller leaves with a higher specific mass. Mexican redbud avoided dehydration by reducing its leaf area while maintaining biomass.

1.5.2 Worldwide species

Cercis species are scattered throughout the north temperate zones of North America and

Eurasia and appear to have become adapted to different climates by altering their leaf morphology, reflecting the environmental conditions and habitats of these plants (Isely

1975, Wunderlin et al. 1981). Cercis canadensis var. canadensis as well as most Chinese species have a dull upper leaf surface and are rather thin leaved, indicating their origin in a humid environment. Cercis canadensis var. mexicana and C. canadensis var. texensis, the western redbud (C. occidentalis), the Mediterranean redbud (C. siliquastrum and the

Afghan redbud (C. griffithii) on the other hand, have thick and leathery leaves. These properties suggest an adaptation to semi-arid or arid environments (Raven 1971,

Wunderlin et al. 1981; Davis et al. 2002).

1.6 Overall objective/hypothesis

Water is the principal factor limiting survival and growth of trees in urban and suburban landscapes (Kramer 1987). Although irrigation can mitigate the impact of drought stress, water restrictions are becoming more common throughout the United States (Cregg

2004). Therefore, selecting trees with improved drought tolerance and reduced water use may be the best strategy to improve survival, growth and health of trees in urban and suburban landscapes. Often trees are selected only for their aesthetic contribution to the landscape, with little consideration towards performance in stressful environments. The variety of plant characteristics that contribute to drought resistance and the variation in site characteristics makes plant selection for dry sites difficult. The aim of this study was to investigate the inter-taxa variation in drought resistance and water use efficiency existing within the genus Cercis, thereby providing information as to their usefulness for plantings in urban landscapes and to facilitate breeding for improved water use and drought resistance. A greater understanding of the physiology, adaptive characteristics, and response to water stress of different species will enhance our ability to select more appropriate species and genotypes for specific sites (Ranney et al 1990).

Variation in growth rate and drought resistance of the Cercis taxa can be explained by variation in patterns of water and carbon acquisition and resource allocation. The interspecific variation has a genetic and an environmental component that results in phenotypic plasticity.

Hypothesis: We hypothesize that C. siliquastrum, C. griffithii and C. occidentalis, (and among the North American sources: C. canadensis var. mexicana, C. canadensis var. texensis and C. canadensis (OK)) are xerix adapted species and that within the North

American species there is a clinal adaptation from xeric to mesic (C. canadensis var. mexicana, C. canadensis var. texensis, C. occidentalis, C. canadensis (OK, OH and PA).

Xeric environment adaptations would include lower shoot-root ratios, a more shrublike growth habit and reduced growth potential (expressed as lower dry weight, smaller LA, smaller leaves, higher root surface area, higher specific root length, higher root area-to- leaf area ratio) have lower water use (per seedling, per unit leaf area and per unit stem height), as well as the ability to maintain higher rates of stomatal conductance and photosynthetic gas exchange during drought and/or recover more rapidly from drought stress and consequently xeric adapted taxa will have higher drought tolerance relative to mesic adapted taxa.

Chapter 2

SPECIES DESCRIPTION

2.1 Abstract

The genus Cercis includes 10 recognized species; however, the taxonomy of Cercis has undergone several changes in concept and the exact number of species remains controversial. The objective of this Chapter was to describe the Cercis taxa used in following experiments (Chapters 3 and 4), to compare them to Cercis described in the literature and describe morphological differences that can influence drought resistance.

Seedling growth habit and leaf characteristics under nursery conditions of seven Cercis taxa (two additional taxa for leaf characteristics) were measured and analyzed using principal component analysis. Taxa described were representative of their species and/or variety. Cercis taxa differed widely in their growth habit and leaf characteristics and showed adaption to their native environments. Some taxa from xeric environments grew tall with few branches and large leaves while other xeric-adapted species were short and shrub-like, with small round leaves. Similarities in growth habit and leaf characteristics were found among taxa that appear unlikely to be closely related. Cercis siliquastrum

(Italy) and the C. occidentalis (California) displayed many similarities in growth habit, and especially in leaf morphology. The persistence of the leaf characteristics under non- limiting soil moisture conditions indicates that the characteristics typical of the taxa grown in their native environment are under genetic control. The fact that some characteristics on the Eastern North American taxa espressed great variability indicates introgression of these charateristics and that the taxa are still in the process of speciation.

Taxa used in this study: C. canadensis (PA, OH, OK), C. canadensis var. texensis, C. canadensis var. mexicana, C. occidentalis, C. griffithii, C. siliquastrum, C. chinensis

Key words: Speciation, leaf morphology, seedling growth

2.2 Introduction

The genus Cercis belongs to the subfamily Caesalpinioideae of the plant family Fabaceae

(Leguminosae). Cercis (Redbud) are trees or shrubs of small to medium size with alternate, undivided, heart- to kidney-shaped, toothless leaves with five to nine conspicuous veins radiating from their base (Everett 1981). Widely cultivated as ornamentals, especially in North America, their reddish to pink or white flowers open before the growth of the leaves and offer a unique view in early spring due to their development on stem or trunk (Coskun 2003). The genus Cercis includes currently10 recognized species scattered widely across the warm, north-temperate zones of North

America and Eurasia. There is some uncertainty as to the exact number of species and their delimitation remains controversial (Fritsch 2009 et al.).

The taxonomy of the Cercis species native to North America is confusing and has undergone several changes in concept. In the traditional concept (Hopkins 1942) the

North American component of the genus Cercis comprised of C. canadensis and C. occidentalis. Cercis occidentalis being restricted to areas west of the Rocky Mountains, chiefly to California, but also locally in Arizona, Utah and Nevada, and C. canadensis.

Cercis canadensis is native throughout the United States and one of the most widely distributed redbuds. The large geographic range of Cercis covers a wide range of climatic conditions. Mean annual precipitation varies from less than 510 mm in south Texas to approximately 1270 mm in central Florida (Dickson 2010). Mean January temperatures vary within the native range of Cercis from -8°C to 16°C. Mean July temperatures from

21°C in southern Pennsylvania to 26°C in central Florida. Associated with this wide

range of climatic and geographic conditions, C. canadensis shows significant clinal variation and dormancy in winter, morphology and hardiness (Donselman 1976,

Donselman and Flint 1982).

Three sub-species/varieties of C. canadensis are recognized: C. canadenis var. canadensis throughout the east of the United States, C. canadensis var. texensis, found in east-central Texas and adjacent Oklahoma, and the Mexican redbud (C. canadensis var. mexicana) found in Trans-Pecos Texas and northeastern Mexico (Hopkins 1942, Correll and Johnston 1979, Turner et al. 2003). Isely (1975) offered four alternatives concerning the recognition of Cercis species in the United States: 1) the traditional classification; 2) there are three species: western United States, Texas/Oklahoma, and eastern United

States (with extensive introgression in the last two as first proposed by Anderson 1953);

3) there are two species, C. canadensis, typically found in more mesophytic habitats and another one including all xerophytic forms (C. occidentalis, C. canadensis var. texensis and C. canadensis var. mexicana) or 4) there is just one species with regional varieties, roughly defined as: Intermountain, northern Californian, southern Californian and the three other traditional varieties of C. canadensis. A study of Salatino (2000) involving flavonoids supports the first alternative, because of the existence of two chemical groups among the North American species of Cercis, where C. canadensis belongs to the group which produces kaempferol and C. occidentalis belongs to another group which is devoid of kaemperol. Davis et al. (2002) conducted a phylogenetic test with ITS and ndhF DNA sequences, which showed that C. occidentalis and C. canadensis were distinct.

Cercis siliquastrum is native to Europe, and five Cercis species (C. chinensis, C. chingii,

C. chuniana, C. glabra and C. racemosa) are indigenous to China (Li 1944, Wodward and Williams 1987, Zou et al. 2008). Cercis griffithiiis native to Afghanistan (Raulston

1968) and described to be very similar to and considered by some an ecotype of C. siliquastrum (Raulston 1990).

The objective of this Chapter was to describe the Cercis taxa to be used in subsequent experiments (Chapters 3 and 4), to compare them to Cercis described in the literature and describe morphological differences that can influence drought resistance. Growth habit and leaf characteristics were measured and principal component analysis employed to describe variation in the observed characteristics.

2.3 Material and methods

In December 2006, seed of the following taxa: Cercis canadensis (OK, 060047), C. chinensis (050748), C. griffithii (010370), C. occidentalis (060033), C. siliquastrum

(050914), C. canadensis var. mexicana (collected at Texas A & M University Research

Center, El Paso, TX) and C. canadensis var. texensis (collected in Dallas, TX, from an isolated planting of trees) were scarified with hot water and stored in a cooler at 4oC until mid-March 2007 when seeds were planted into flats filled with media (Fafard 3B, Conrad

Fafard, Inc., Agawam, MA) and placed on greenhouse benches. All seeds except the

Ohio source were obtained from Sheffield’s Seeds Company, Inc. (Locke, NY; seed lot numbers are given in parenthesis).

In April 2007 the seedlings were transplanted into containers (13 cm square x 15 cm deep, 1.97 L, Classic 250, Nursery Supplies, Chambersburg, PA) filled with media 40

(Fafard 25, Conrad Fafard, Inc., Agawam, MA) and grown pot-to-pot in the greenhouse.

Seedlings were fertigated with 20-10-20 Plantex High Nitrate (Plant Products Co. Ltd.

Canada), 100 g N twice a week, starting two weeks after transplantation. In October 2007 plants were moved to a minimum heat polyhouse (1 to 3 C) for overwintering. In spring

2008 plants were placed in No. 3 containers (11.4 L, Classic 1000, Nursery Supplies,

Chambersburg, PA) filled with a 3:1 Pine bark:Comtil (City of Columbus, OH) Mix. In

May 2008, plants were lined out in a completely random pattern on a gravel container pad (0.5 m within row and 1.0 m between row spacing) in full sun at The Ohio State

University Waterman Farm (Columbus, OH). Plants were fertilized with 45 g of 18N-

2.6P-7.8K Osmocote fertilizer per container and irrigated twice a day via micro-irrigation at one L per irrigation event. Plants were allowed to grow without additional training or pruning. During the winter of 2008/2009 plants were overwintered and grown in a similar manner as in the first growing season. In fall 2009, the plants were measured using the methods described below. Two additional taxa were included in the leaf description, C. canadensis (PA) and C. canadensis (OH) but, these taxa were excluded from the other measurements as they were 1-yr old while the seedlings of the other taxa were 2-yr old.

2.3.1 Growth habit

Ten plants per taxa were measured for the length of the main shoot axis and the number and length of first, second, third and fourth order branches. The numbers of nodes were counted as well as the height of the point of the attachment of the first branch to the central axis. The first branch height was expressed as the percentage of the total plant height by dividing the branch attachment height by the total plant height.

2.3.2 Leaf morphology

Three mature leaves from five plants of each taxon were used for the description of leaf character measurements. The characters examined were: leaf blade length 1 (measured from the apex to the base of the midvein), leaf blade length 2 (measured from the apex to the bottom of the basal lobe), leaf blade width (measured at the point of maximum width perpendicular to the midvein), leaf ape (estimated in degrees: degrees acuminate

(1), degrees sub-acuminate (2), > 45 degrees = acute (3), > 90 degrees = obtuse

(4), round (5) (Dirr 1998), basal sinus width (measured width at midpoint of sinus depth), basal sinus depth: (from bottom of basal lobe to bottom of midvein), number of main veins, pubescence of petiole and abaxial surface (present or absent), petiole length

(measurement included distal pulvini to base of leaf), petiole width (measured width at center of petiole, color of petiole and abaxial veins (green, greenish-red, red), glaucous

(presence of absence of waxy bloom on abaxial leaf surface) (Fig. 2.1). Leaf blade width

2 and leaf width were used to calculate the leaf blade ratio (leaf blade width 2/ leaf blade width) as an indicator of leaf shape. A lower number represented an ovate leaf while a higher number represented a round or oblate leaf shape. The basal sinus area was calculated by multiplying half of the basal sinus width with the basal sinus length. The characters petiole pubescence and abaxial surface pubescence as well as an undulate leaf margin were found to co-vary completely and were combined into one parameter called pubescence. The characters of leaf blade length (1, 2), leaf blade width, basal sinus width and length, number of veins, petiole length and width were continuous variables; leaf blade margin, pubescence on petiole and abaxial surface were binary. Leaf apex was a categorical variable.

Statistics

A principal component analysis was performed using PROC FACTOR with varimax rotation in SAS 9.2 (SAS Institute, Cary, NC). The analysis was performed separately for growth habit and leaf description, using thirteen and twelve variables, respectably. The first three principal components were plotted in a 3D scatter plot. Data was also subjected to an analysis of variance using the generalized linear model (PROC GLM) of SAS 9.2 using 10 single plant replications for growth characteristics and 15 single plant replications for leaf characteristics, with species as the independent factor. When significant species differences were found, means were separated by using the Waller-

Duncan test at P< 0.05.

Leaf length 1 Leaf length 2

Leaf width Main vein

A Basal length A B B Basal width

Petiole length Petiole width

Fig. 2.1 Description of measurements taken on mature leaves of Cercis L.

2.4 Results

2.4.1 Growth habit

The first three principal components explained 71% of the total variance (Table 2.1). The first principal component (PC1) accounted for 47% of the total variance and was considered as a surrogate for branching habit. The number of second, third and fourth order branches as well as the average length of third and fourth order branches were highly and positively correlated with the first component. Total shoot length, a combination of height and length all branches of all orders, was also highly positively correlated.

The second principal component (PC2) accounted for 14% of the total data set variance

(Table 2.1) and was considered as a surrogate for plant growth habit. Caliper, height, the number of nodes on the main axis, and the position of first branch attachment on the main axis were all highly and positively correlated with PC2. Plants with larger caliper and those having the point of attachment of the first branch higher on the main axis tended to load positively on the PC2 axis.

The third principal component (PC3) accounted for 10% of the total variance (Table 2.1) and was a surrogate for the initial branching habit of the plants. The length of first and second order branches were positively correlated while the number of first order branches was negatively correlated with PC3. Plants loading positively on PC3 tended to have either few but long first and second order branches, or many short ones.

Variable PC 1 PC 2 PC 3 Caliper 0.14 0.63 0.05 Height -0.46 0.78 -0.01 No. of nodes -0.42 0.68 0.05 1st order branches Number 0.63 -0.11 -0.67 Average length -0.13 -0.18 0.85 2nd order branches Number 0.84 -0.18 0.01 Average length 0.32 0.10 0.62 3rd order branches Number 0.84 -0.13 -0.05 Average length 0.71 -0.40 0.09 4th order branches Number 0.85 -0.15 -0.21 Average length 0.86 -0.26 0.21 Total shoot length 0.84 -0.07 0.18 First branch height -0.44 0.63 -0.10 Cumulative percent of total variation explained 47 61 71

Table 2.1 Rotated factor pattern for principal component analysis of growth characteristics of seven Cercis L. taxa.

The separation of C. canadensis var. mexicana from the other taxa on the PC1 axis (Fig.

2.2) was due to its large number of higher order branches and consequently the large total shoot length. Cercis chinensis and C. siliquastrum clustered on PC1 and PC3 due to similarities in number and length of higher and lower order branches which were also similar to C. canadensis var. texensis (Table 2.2). The separation among these three taxa on the PC2 axis was due to significant greater height and caliper of C. chinensis, as well as the greater height of the first branch as percentage of its total height relative to C. siliquastrum (Table 2.2). Cercis occidentalis was adjacent to C. griffithii in all three PC axes. However, C. occidentalis had significantly longer third and fourth order branches,

and the point of attachment of the first branch as percent of the total plant height was higher (PC1) than in C. griffithii (Tables 2.2 and 2.3). Cercis griffithii and C. siliquastrum showed few differences in characteristics portrayed in PC1 and only differ in the number of nodes on the main shoot axis on PC2 (Table 2.2). However, the significant difference in the length of the first order branches separated both species on the PC3 axis. Cercis canadensis var. texensis and C. canadensis (OK) showed no difference in any of the variables observed in the PC a is’s (Tables 2.2 and 2.3), but the combination of characters showed separation on PC2 and PC3 axes.

The taxa differed significantly in caliper (Table 2.2). Cercis canadensis (OK), C. canadensis var. texensis and C. chinensis had the greatest caliper, while C. occidentalis had the smallest. The taxa C. canadensis (OK) and C. canadensis var. texensis did not differ significantly in any of the observed characteristics. Strong similarities were observed between C. siliquastrum and C. griffithii, which differed only in the average length of their first order branches (Table 2.3). Cercis chinensis, was one of the tallest taxa and had highest number of nodes on the main shoot axis (Table 2.2). Cercis canadensis (OK) and C. canadensis var. texensis had no third or fourth order branches

(Table 2.3). Cercis griffithii had the longest first order branches, while C. siliquastrum had the shortest. Cercis canadensis var. mexicana had the highest total shoot length, attributed to the large number of second, third and fourth order branches, rather than its main shoot axis height. Cercis chinensis had the lowest total shoot length based on its low number on second order branches and the complete lack of third and fourth order branches (Table 2.3). The height of the first branch, as percentage of the main axis height

was lowest in C. occidentalis and C. canadensis var. mexicana; and greatest in C. chinensis.

0.6 C E 0.4 . . I c 0.2 c Ga a n 0.0 .

PC 3 PC n F D c . . . -0.2 . a v v c c n a -0.4 a r a 2.5 a . 2.0 r . n -0.6 G n v 1.5 . t .1.0 . . H a -0.8 m 0.5v 1.0 . e 0.8 c v r 0.0 PC 1 0.6e 0.4 x a 0.2 a c . a 0.0 -0.5 x -0.2 r -0.4 a e n r -0.6 t -1.0 i PC 2 -0.8n . . . n e c s t v t . x a i e a e v e n s x r x a n a e . e r s n t n . i Fig. 2.2 Plotted values of the first three principal components ofs the growth habit e s t s description of Cercis L. taxa. C= C. occidentalis, D= C. canadensisi (OK), x i e E= C. canadensis var. texensis, F= C. canadensis var. mexicanas , G= C. chinensis, e s x H= C. siliquastrum, I= C. griffithii. n e s n i s s i s 48

Taxa Caliper Height Nodes Total shoot length Height of first branch (mm) (cm) (no.) (cm) (%) C. occidentalis 13.5y ez 167 b 46.4 bc 1165 b 3.3 d C. canadensis (OK) 20.7 a 242 a 46.7 bc 886 bcd 40.3 b C. canadensis var. texensis 19.6 ab 246 a 52.3 b 945 bc 40.1 b C. canadensis var. mexicana 16.6 cd 109 c 32.5 d 1755 a 2.7 d C. chinensis 19.4 ab 236 a 64.3 a 631 d 50.8 a C. siliquastrum 15.7 d 184 b 51.3 b 675 cd 16.4 c C. griffithii 18.4 bc 182 b 38.0 cd 961 bc 18.4 c

4 P > F <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Table 2.2 Caliper, height and shoot growth of seven Cercis L. taxa.

y Each value is the mean of 10 plants per taxon.

z Means within a column followed by different letters are significantly different from each other at α 0.0 using the Waller- Duncan test of significance.

Second, third and fourth order Taxa First order branches branches No. Length No. Length C. occidentalis 13.9y bz 44.4 ab 25.8 b 29.1 b C. canadensis (OK) 13.8 b 40.7 abc 7.4 c 14.8 cd C. canadensis var. texensis 13.1 b 40.1 abc 7.7 c 23.5 bc C. canadensis var. mexicana 24.0 a 33.2 bc 76.6 a 46.7 a C. chinensis 11.6 b 31.6 cd 1.2 c 9.1 d C. siliquastrum 13.1 b 28.2 d 10.7 c 13.7 cd C. griffithii 12.3 b 48.4 a 11 c 18.1 cd

50 P > F 0.0008 0.0045 <0.0001 <0.0001

Table 2.3 Branching habit of seven Cercis L. taxa.

y Each value is the mean of 10 plants per taxon.

z Means within a column followed by different letters are significantly different from each other at α 0.0 level using the Waller-Duncan test of significance.

2.4.2 Leaf morphology

The first three principle components accounted for 73% of the total variance (Table 2.4).

PC1 accounted for 50% of the total variance. Leaf blade length (apex to petiole), leaf blade width, basal sinus width and area as well as petiole length and width were highly and positively correlated with PC1. Apex angle and glaucousness were highly and negatively correlated with the PC1. PC1 represented the basic morphological structure of the leaves. It showed the trend that larger leaves (long and wide, wide basal sinus and large basal area, and long and thick petioles) have a tendency towards more acuminate type leaf apices and glabrous adaxial leaf surfaces.

The PC2 accounted for 14% of the total variance in the data (Table 2.4). Leaf ratio (as a measure of leaf shape) and the color of the petiole were positively correlated with the

PC2. PC2 showed that ovate leaves with a (higher leaf ratio) tended to have a more reddish petiole color, while leaves with a more round leaf shape had predominantly green petioles.

PC3 accounted for 9% of the total variance observed (Table 2.4). The number of leaf veins and the pubescence of petiole and abaxial surface of the leaf were positively correlated with PC3. Leaves with a higher number of veins tend to have pubescent petioles and abaxial leaf surfaces, and undulate leaf margins.

Variable PC 1 PC 2 PC 3 Leaf blade length 1 0.96 0.08 -0.12 Leaf blade width 0.93 -0.20 -0.05 Leaf blade ratio 0.38 0.73 -0.07 Apex angle -0.72 -0.39 -0.02 Basal sinus width 0.91 0.02 -0.04 Basal sinus area 0.78 -0.25 0.15 Petiole & vein color -0.19 0.68 -0.08 Glaucousness -0.84 -0.18 0.06 No. of veins 0.15 -0.32 0.81 Pubescence 0.49 0.43 0.62 Petiole length 0.69 -0.11 -0.35 Petiole width 0.80 -0.03 0.06 Cumulative percent of total variation explained 50 64 73

Table 2.4 Rotated factor pattern for principal component analysis of leaf characteristics of seven Cercis L. taxa. Loadings above 0.60 are shown in bold.

Cercis canadensis var. mexicana showed distinct separation from all other taxa (Fig.2.3).

It shared some common features with C. occidentalis, but had significantly narrower leaves as well as a less obtuse leaf apex (Table 2.5). Cercis canadensis var. mexicana showed similar values to C. chinensis on the PC2 axis with regards to having a relative ovate leaf shape (Table 2.5) and a reddish petiole and vein color (Table 2.6).

Cercis occidentalis and C. siliquastrum clustered together (Fig. 2.3), but showed similarities on the PC1 axis only in the round leaf apex, the fairly small basal sinus area and the glaucous adaxial leaf surface (Tables 2.5 and 2.6). On the PC2 axis the leaf shape ratio was greater in C. siliquastrum than C. occidentalis, which indicated that its leaves were more ovate while the leaf shape of C. occidentalis was more rounded.

Cercis canadensis (PA), C. canadensis (OH) and C. canadensis var. texensis formed a cluster in the three dimensional space (Fig. 2.3). Cercis canadensis (PA) and C. 52

canadensis (OH) grouped together on PC1; both possessed wide leaves, a large basal area, had glaborous adaxial leaf surfaces (Table 2.4.a) and relatively long and thick petioles (Table 2.5). Cercis canadensis (PA), C. canadensis (OH) and C. canadensis var. texensis showed no differences in petiole/vein color, however, C. canadensis var. texensis had a significantly smaller leaf shape ratio than C. canadensis (PA), making its leaves more broadly ovate (Table 2.5). Leaves of C. canadensis (PA) showed a significantly lower number of veins per leaf (Table 2.6) than the two other taxa included in this group, which explained its slightly lower value on the PC3 axis.

Cercis canadensis (OK), C. chinensis and C. griffithii clustered together (Fig. 2.3).

However, at least two of the three taxa differed significantly for any leaf characteristic loading on PC1. Leaf shape ratio, which loaded on PC2, showed no difference between the three taxa and categorized the leaves as the most ovate of all taxa (Table 2.5). None of the taxa displayed pubescence on petiole or veins, therefore, the slightly lower value of

C. chinensis on the PC3 axis was attributed to the significant lower number of veins compared to C. canadensis (OK) and C. griffithii (Table 2.6).

The taxa showed significant differences (P<0.0001) in examined leaf characters.

Differences between taxa were greater for leaf blade length 1, than for leaf blade length 2

(Table 2.5). However, Cercis canadensis (PA) had the highest values for leaf blade length1 and 2, while C. occidentalis and C. canadensis var. mexicana had the shortest.

Leaf blade width was largest in the northern sources of C. canadensis from Pennsylvania

(PA) and Ohio (OH). Cercis canadensis var. mexicana had the narrowest leaf blade.

Cercis canadensis (PA), (OH) and (OK), C. canadensis var. mexicana, C. chinensis and

C. griffithii had a similar leaf blade ratio (Table 2.5), indicating a more ovate leaf shape, with a leaf blade length greater than its width. In contrast, C. occidentalis and C. siliquastrum had a leaf blade ratio less than one, indicating an oblate leaf shape. Cercis canadensis (PA) had the widest sinus width and as a result one of the largest basal sinus areas (Table 2.6). The shortest basal sinus width and lowest basal sinus area were observed for C. occidentalis and C. canadensis var. mexicana. Cercis canadensis (OK) showed relatively short basal sinus length for the associated width. Cercis canadensis

(PA) and (OH), and C. siliquastrum displayed fully green petiole and abaxial vein color

(Table 2.6). Cercis canadensis var. mexicana, C. chinensis and C. griffithii leaves veins were reddish-green. In C. canadensis var. mexicana this was the result of actual reddish- green vein color; however, in both of the other taxa, the reddish-green average was a result of some leaves displaying green petioles and veins while others displayed red ones.

Glaucous adaxial surfaces were found for C. occidentalis, C. canadensis var. texensis and

C. siliquastrum (Table 2.6). Cercis canadensis (OH) and C. canadensis var. texensis had the most main veins while C. chinensis had the least (Table 2.6). Cercis canadensis var. mexicana was the only taxa that displayed undulate leaf margins and pubescence on petioles and the abaxial leaf surface (Table 2.6). Petiole length was shortest in C. canadensis var. mexicana, which also had a relatively narrow width. Cercis griffithii displayed the longest petioles, while C. canadensis (PA) and C. chinensis had the widest petioles (Table 2.6).

2.0 F 1.5 B A 1.0 E D

0.5 PC3

0.0 G I

-0.5 1.5 1.0 -1.0 H 0.5 C 0.0 -1.5 -0.5 1.0 0.5 -1.0 PC1 0.0 -1.5 -0.5 PC2 -1.0 -2.0 -1.5

Fig. 2.3 Plotted values for the first three principal components of the leaf description of Cercis L. taxa. A= C. canadensis (PA), B= C. canadensis (OH), C= C. occidentalis, D= C. canadensis (OK), E= C. canadensis var. texensis, F= C. canadensis var. mexicana, G= C. chinensis, H= C. siliquastrum, I= C. griffithii.

Leaf blade Taxa Length 1 Length 2 Width Ratio Apex Glaucousness (adaxial surface) (mm) (mm) (mm) C. can. (PA) 138y az 158 a 153 a 1.03 a 2.1 e 1.0 c C. can. (OH) 131 b 157 a 155 a 1.01 ab 2.9 c 1.0 c C. occidentalis 57 g 76 f 85 f 0.89 d 4.7 a 2.0 a C. can. (OK) 106 d 118 cd 115 cd 1.03 a 1.9 e 1.1 b C. can. var. texensis 95 e 110 d 113 d 0.98 bc 2.6 cd 1.0 c C. can. var. mexicana 56 g 72 f 71 g 1.01 ab 3.9 b 2.0 a

56 C. chinensis 124 c 143 b 139 b 1.04 a 2.3 de 1.1 bc

C. siliquastrum 75 f 95 e 103 e 0.94 c 4.5 a 2.0 a C. griffithii 108 d 127 c 124 c 1.03 a 2.7 c 1.0 c

P > F <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Table 2.5 Lamina characteristics of nine Cercis L. taxa.

y Each value is the mean of 15 leaves per taxon.

z Means within a column followed by different letters are significantly different from each other at α 0.0 using the Waller- Duncan test of significance.

Basal sinus Veins Petiole Pubescence, undulate leaf margin Taxa Length Width Area Length Width (mm) (mm) (mm) (no.) (mm) (mm) C. can. (PA) 21y bz 66 a 691 a 8.1 b 45 bc 1.8 a 1 b C. can. (OH) 27 a 55 b 732 a 8.9 a 48 ab 1.7 ab 1 b C. occidentalis 18 bc 27 f 242 cd 7.1 c 34 g 1.2 f 1 b C. can. (OK) 10 d 54 bc 291 cd 7.3 c 43 cd 1.6 bc 1 b C. can. var. texensis 15 c 46 d 359 c 8.9 a 36 fg 1.5 cd 1 b C. can. var. mexicana 15 c 27 f 205 d 8.1 b 27 h 1.3 ef 2 a

C. chinensis 19 b 56 b 548 b 5.9 d 40 de 1.8 a 1 b C. siliquastrum 20 b 34 e 345 c 7.0 c 39 ef 1.4 de 1 b C. griffithii 19 b 48 cd 493 b 7.0 c 50 a 1.6 bc 1 b

P > F <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Table 2.6 Leaf sinus, vein, petiole and pubescence characters for nine Cercis L. taxa.

y Each value is the mean of 15 leaves per taxon.

z Means within a column followed by different letters are significantly different from each other at α 0.0 using the Waller- Duncan test of significance. 57

2.5 Discussion

Cercis taxa described here show characteristics typical for the taxa as described in the literature. However, oftentimes the literature is inconclusive on certain characteristics of some taxa and some taxa described here showed a great variation in certain characteristics.

Principal component analyses were employed to detect relationships among the taxa based on plant growth and leaf morphological characteristics. There appears to be no clinal variation among the North American taxa under the conditions of this experiment.

Clinal variation among the North American taxa appears to be unlikely based on plant growth characteristics. Although C. canadensis (OK) and C. canadensis var. texensis load similarly on PC1 (branching habit), C. canadensis var. mexicana was separated from their cluster. Based on plant growth characteristics only two taxa clustered together, C. griffithii and C. occidentalis, not unexpected as both occur in Mediterranean environments and represent apossible example of convergent evolution.

The principal component analysis based on leaf morphology revealed three clusters and a single outlying taxon. Cercis canadensis (OK), C. chinensis and C. griffithii appeared in the same cluster, even though C. canadensis (OK) and C. griffithii are considered xeric adapted species and C. chinensis is considered a mesic adapted species. Cercis occidentalis and C. siliquastrum formed a second cluster and three North American taxa

(C. canadensis (PA and OH) and C. canadensis var. texensis) formed a third cluster.

Cercis canadensis var. mexicana was separate from the all other clusters.

Although, C. canadensis (PA, OH) and C. canadensis var. texensis cluster together, three other North American taxa (C. occidentalis, C. canadensis (OK) and C. canadensis var. mexicana) are not included in that cluster, making clinal variation among North

American taxa unlikely. Cercis occidentalis and C. siliquastrum clustered together, even though their origins are geographicly isolated. Cercis siliquastrum is native to the

Mediterranean where dry summers and rainy winter prevail. However, this Mediterranean climate does not only occur in the Mediterranean region but also in the native range of C. occidentalis (California). The expression of similar characteristics in allopatric species suggests convergent evolution.

Some taxa adapted to xeric environments displayed some hypothesized growth habits and leaf characteristics, while others did not. Cercis occidentalis, C. canadensis var. mexicana, Cercis griffithii and C. siliquastrum, i.e. displayed the typical shruby growth habit of xeric adapted species as evident by their clustering on PC2 (plant growth characteristics). However, in the principal component analysis of leaf morphology C. griffithii clustered with C. canadensis (OK) and C. chinensis, were C. chinensis is considered a mesic adapted species. This grouping is primarily based on the loadings on

PC1 (basic leaf morphology) including leaf blade length and width and apex shape. It should be noted that these morphologies were expressed in juvenile seedling grown under non-limiting soil moisture conditions.

Cercis canadensis

Cercis canadensis are small to medium sized trees that can reach up to 12 m tall. They are described as the hardiest and most tree-like looking of the Cercis species, although the trunk is often divided close to the ground forming a spreading and rounded crown

(Raulston 1968, Dirr 1998). Their simple leaves are 7.5-12 cm long and of similar or greater width (Dirr 1998). The broad ovate to broadly heart-shaped leaves have five to nine radiating veins and are pubescent to glabrous on the adaxial leaf surface. Dirr (1998) describes the leaf apex as acute and the basal sinus as cordate. The leaf petiole has a pulvinus just below the leaf blade and varies in length from 3-6 cm. Dirr (1998) list is as hardy in the Hardiness Zones 4 to 9.

The here described sources of C. canadensis from Pennsylvania, Ohio and Oklahoma showed similarities in leaf characteristics. Cercis canadensis from Pennsylvania (Fig. 2.5

H) and Ohio (Fig. 2.5 I) were most alike, although C. canadensis (OH) had a mostly cuspidate leaf apex while the Pennsylvanian source showed a more acute leaf tip, similar to C. canadensis (OK, Fig. 2.5 J). Leaves of C. canadensis (OK) were smaller than the two other C. canadensis sources. Differences were also significant for the basal sinus, C. canadensis (OK) had a much shorter basal sinus by comparable width, leading to a smaller basal sinus area. The number of main veins was greatest in C. canadensis from

Ohio and lowest for C. canadensis (OK); for C. canadensis from Pennsylvania both leaves with seven and nine veins were observed.

Cercis canadensis var. texensis

Cercis canadensis var. texensis is a small tree, usually 5-7 m tall (even up to 13 m) with a rounded or vase shaped growth habit (Hopkins 1942). Hopkins (1942) described the plant as similar to C. canadensis when growing in the field. Only the leaves differ greatly by being relatively thick, leathery and glaucous with a typical sheen while leaves of C. canadensis are merely dull green. Kruessmann (1984), however, describes the plant as always multi-stemmed from the base and often only 1-2 m tall. The leaves are kidney- shaped, 5-7 cm wide and glossy on the adaxial surface.

The C. canadensis var. texensis (Fig. 2.4 B) described had a similar growth habit to C. canadensis (OK, Fig. 2.3 A). No significant differences in the described growth characteristics were observed. However, although leaf blade length 2 and leaf blade width were not significantly different, the leaf blade ratio described the leaves of C. canadensis var. texensis as slightly oblate, while leaves of C. canadensis (OK) were slightly longer than wide. Differences in the leaf apex were also observed, C. canadensis (OK) displayed, on average, a cuspidate apex, while the apex of C. canadensis var. texensis were slightly wider and more acute. Cercis canadensis var. texensis has a typical glossy sheen on the adaxial leaf surface was observed in some plants grown for the experiments

(chapter 3 and 4), but not in plants described in this study. The difference in number of main veins per leaf was especially significant, with the leaves of C. canadensis (OK) almost exclusively displaying seven veins, while leaves of C. canadensis var. texensis showed mostly nine. The leaf blades of both northern sources of C. canadensis (Ohio and

Pennsylvania, Fig. 2.5 I and H, respectively) were significantly longer and wider than leaves of C. canadensis (OK) and C. canadensis var. texensis, and in shape more similar 61

to the southern source of C. canadensis from Oklahoma (Fig. 2.5 J) than to C. canadensis var. texensis. However, the leaf apex of the Ohioan source was mostly acute, while the source from Pennsylvania displayed shapes from acuminate to acute.

Cercis canadensis var. mexicana

The mostly multi-stemmed Cercis canadensis var. mexicana can grow into a 2 m tall shrub or a 4 m tall tree (Ladybird Johnson Wildflower Center 2010, Raulston 1990). The plants are densely branched (Raulston 1968) with a crown that is rounded or vase shaped but irregular in outline (Gilman and Watson 1993). Leaves are small with varying sizes reported. Raulston (1968) reports leaf sizes of 2.5 to 15 cm, while Dirr (1998) reports sizes from 5 to 7.5 cm. The leaves of C. canadensis var. mexicana have undulate margins

(Raulston 1968, Dirr 1998). The foliage is extremely variable, with either a thin, dull cuticle or a thick and glossy one (Tipton and White 1995). Leaves of C. canadensis var. mexicana have been described as cordate, orbiculate or ovate, with a leaf blade length of

5-10 cm (Gilman and Watson 1993). Young branches and petioles are covered with a brownish, very tomentose pubescence (Hopkins 1942). Dirr (1998) reports great variation in this taxon, describing plants with a large statue and large, minimally glossy and undulating margined leaves.

Cercis canadensis var. mexicana described here concur with descriptions in the literature

(Raulston 1968, Gilman and Watson 1993, Tipton and White 1995, Dirr 1998). Plants were mostly shrub-like and densely branched with small leaves with undulate margins and obtuse apex. The adaxial leaves surfaces of the here described plants were glaucous, although single plants with glossy leaves were observed in the population. As described

by Hopkins (1942) young branches, petioles and abaxial leaf surfaces were pubescent.

The population grown from seed showed a great variation in both growth and leaf characteristics. Sporadically, plants with large, non-undulate or glossy leaves were observed, as well as plants that were more tree-like and similar to C. canadensis.

Cercis occidentalis

Cercis occidentalis usually grows as a spreading, multi-stemmed shrub with a dense crown that almost reaches the ground (Brickell and Cathey 2004, National Resources

Conservation Center 2010), but can reach tree-size in some locations (Kruessmann 1984,

Raulston 1990). Plants with heights of 1 m as well as 3-6 m have been described in plants growing in the native range of this species. It leaves are simple and rounded or heart shaped and have seven to nine main veins (National Resources Conservation Center

2010). The small leaves are only 2.5 to 7.5 cm in diameter, often obtuse (wider than long) and glaucous with a notched or round leaf apex (Kruessmann 1984, Dirr 1998).

Cercis occidentalis described here, were multi-stemmed trees of medium height compared to other taxa (Fig. 2.4.D). A large number of first, second and third order branches and the low attachment of the first branch on the main shoot axis results in a spreading crown shape. This highly branching shrub-like growth habit with a very low point of attachment of the first branch is similar to C. canadensis var. mexicana habit, although this taxon was more densely branched with fourth order branches, which were not observed for C. occidentalis (Fig. 2.4 D). Leaves of both taxa were also similar in size, however the leaf blade ratio indicated an oblate shape for C. occidentalis (Fig. 2.5

K) and a round shape for C. canadensis var. mexicana. Similarities were also found in

leaf apex with an obtuse apex for C. occidentalis and an obtuse to round apex for C. canadensis var. mexicana. A glaucous adaxial leaf surface was observed in both species.

Basal sinus areas were similar in length and width. Leaf petioles were longer in C. occidentalis. Most stricing differences are the pubescence and undulate leaf margins existend in C. canadensis var. mexicana but lacking in C. occidentalis.

Cercis siliquastrum

Cercis siliquastrum are spreading, sometimes multi-stemmed trees that grow up to 12 m in height in cultivation; however, most plats do not exceed a height of 5 to 7 m (Raulston

1990). Their heart-shaped leaves are 5-10 cm high and up to 12.5 cm wide, heart-shaped at the base with a rounded or notched tip and usually seven main veins (Kruessmann

1984, Raulston 1990, Dirr 1998).

The species is very variable and there seems to be a great ecotypic variation in nature as well as in cultivation and many of these variants have been recognized as either different species or varieties (Robertson 1976, Raulston 1990).

Cercis siliquastrum observed here were mostly multi-stemmed trees with a relative large number of first and second order branches (Fig. 2.4 E). Its growth habit was very similar to C. occidentalis Fig. 2.4 D), differing only in a higher attachment point of the first branch and fewer third and fourth order branches in C. siliquastrum. Leaves of C. siliquastrum (2.5 N) were larger than those of C. occidentalis (Fig. 2.5 K); however, differences in other observed leaf characteristics were minimal. Both taxa displayed glaucous adaxial surfaces.

Cercis chinensis

Cercis chinensis is considered the tallest redbud and grow strictly upright. In the wild they can reach a height up to 17 m and a trunk caliper of 0.9 to 1.2 m. In cultivation C. chinensis tends to be a multi-stemmed small shrub or tree, reaching only 3-5 m in height

(Raulston 1968, Robertson 1976, Everett 1981). The leaves have rounded, 7- 12 cm long and almost as wide, with acuminate leaf apex and a deeply cordate base (Kruessmann

1984).

Cercis chinensis (Fig. 2.4 F) described here tall and in growth habit similar to C. canadensis (OK, Fig. 2.4 A) and C. canadensis var. texensis (Fig. 2,4 B). However, C. chinensis was relatively poorly branched compared to the two other taxa. Leaves of C. chinensis (Fig.2.5 M) were more similar to C. griffithii (Fig. 2.5 O) although apex shape in C. chinensis was slightly more cuspidate while C. griffithii showed a more acute tip.

Cercis griffithii

Raulston (1968) described C. griffithii it to be very similar to and considered it an ecotype of C. siliquastrum (“Cercis griffithii”, IUCN 2011), although, the here described

C. griffithii (Fig. 2.4 G) show similarities in growth habit, leaf characteristics do not support Raulston’s (1968) consideration of C. griffithii being an ecotype of C. siliquastrum. Cercis griffithii (Fig. 2.5 O) described here had larger leaves than C. siliquastrum (Fig. 2.5 N) and were ovate shape while the leaves of C. siliquastrum were usually oblate. Leaf apex shape differed significantly between the two species, with C. griffithii having a mostly acute tip, where C. siliquastrums apex is optuse to round. The

C. siliquastrum typical glaucousness on the adaxial leaf suface was not observed in C.

griffithii. Althought similar in basal sinus length, C. griffithii had a significantly wider basal sinus, resulting in a larger basal sinus area. However, both taxa had seven main veins per leaf.

A. B. C. C. canadensis (OK) C. canadensis var. C. canadensis var. texensis mexicana

Fig. 2.4 A-G. Photographs of one-year old plants of Cercis L. taxa. The background in all pictures is 2 m tall.

Continued 66

Fig. 2.4. continued

D. E. F. C. occidentalis C. occidentalis C. siliquastrum C. chinensis

Continued

Fig. 2.4. continued

C. griffithii

a b

H. C. canadensis (PA)

a b

I. C. canadensis (OH)

Fig. 2.5 H-O. Photographs of (a) adaxial and (b) abaxial leaf surface of Cercis L. taxa.

Continued 69

Fig. 2.5. continued

a b

J. C. canadensis (OK)

a b

K. C. occidentalis

a b

L. C. canadensis var. texensis Continued

Fig. 2.5. continued

a b

M. C. chinensis

a b

N. C. siliquastrum

a b

O. C. griffithii

Donselman and Flint (1982) observed that leaves of plants of C. canadensis from a southern origin had smaller leaves that those of northern origin, even under identical light conditions. Fritsch et al. (2009) observed a clinal adaptation to regional precipitions regimes in Cercis. Leaf size decreased and leaf blade apex shape became more rounded as precipitation decreased. Our results support these findings; taxa from xeric environments (C. occidentalis (California), C. canadensis var. mexicana (Texas), and C. canadensis var. texensis) had smaller leaves and a more rounded leaf apex than the taxa from mesic environments. Lack of water frequently limits plant growth under high light conditions. While larger leaves would help maximize light capture under shaded conditions, the interception of high solar radiation in dry areas has no adaptive advantage and may have presented a selection pressure for smaller leaves (Donselman and Flint

1982, Fritsch et al. 2009). Donselman and Flint (1982) observed that western populations of C. canadensis had blunter leaf tips and were more rounded than leaves from eastern populations. The reduction in size of plant parts has often been observed in drought adapted plants. The rounded leaf apices minimize the surface-to-volume ratio, thereby reducing evapotranspiration assuming no other concommitent changes in morphology

(Donselman and Flint 1982, Fritsch et al. 2009). Donselman and Flint (1982) found acuminate leaf apices in redbuds of northern and eastern origin. Acuminate leaf apices

(drip tips) are thought to prevent growth of epiphyllus microorganisms by allowing runoff of dew and accumulated rain fall from the leaf (Dean and Smith 1978, Donselman and Flint 1982). However, the adaptive significance of driptips has not been resolved

(Baker-Brosh and Peet 1997).

The absence of acuminate leaf apices, and the lack of water shedding may provide another advantage for redbuds in xeric areas since dew lowers the leaf temperature on the following morning, reducing evaporation and water use. Basal sinuses were observed to be shallower in shouthern populations, but no adaptive value was apparent (Donselman and Flint 1982).

Glaucousness, a waxy substance covering the cuticle, was observed in C. occidentalis and C. siliquastrum. Both species are native to xeric environments with high light, high temperature conditions. Glaucousness increased drought resistance reduced cuticular water loss (Quariani et al. 2000) and increased reflection of incoming radiation at the UV and 400-700 nm wavelength to the extent that transpiration and leaf temperature were reduced without a reduction in stomatal conductance (Blum 2005). Genetic differences have been identified by Nagarajah (1979), suggesting that cuticular effectiveness could be improved through selection and breeding. However, this trait is beneficial under drought, but reflective properties come at a price during times of sufficient water availability. They are often associated with reduced photosynthesis and ultimately slower growth (Sanchez et al. 2001).

The Cercis taxa described here varied widely in their plant growth characteristics. Some taxa, like C. chinensis grew tall and developed only a few, long first order branches.

Other taxa, like C. canadensis var. mexicana or C. occidentalis, developed a shrub-like growth habit, with branches of several orders. Plant branch architecture influences stem flow; the portion of precipitation that is intercepted by the vegetation cover and re- directed down the plants branches and stems. Through this process, the ground area

around the plants stem receives additional moisture (Steinbuck 2002) and could be considered as a xeric-site adaptation.

Crockford and Richardson (2000) documented that stemflow accounts for as much as 5 to

10% of the incident gross precipitation. However, stemflow is of importance to the plant since it provides spatially localized input of water. The volume of stemflow is influenced by in part by precipitation intensity, crown morphology, and stem morphology

(Steinbuck 2002). Tree morphology, especially crown area is a significant factor determining the amount of stemflow (Lawson 1967, Aboal et al. 1999). Trees with large crowns have a greater capacity to capture precipition than trees with smaller crowns

(Steinbuck 2002) and to concentrate moisture within its root zone.

Plant height and leaf area were highly correlated. Rapid growth rate would be an adaptive advantage in a mesic environment where soil moisture is less limiting than light interception. However, rapid growth rate in a xeric environment might result in death.

The similarity of the leaf characteristics under non-limiting soil moisture indicated that the characteristics typical of the taxa grown in their native environment are under genetic control. The observation that some characteristics on the Eastern North American taxa espressed great variability indicates introgression of these charateristics and that the taxa might still in the process of speciation.

Chapter 3

WATER USE AND MORPHOLOGY OF EIGHT CERCIS TAXA

- INSIGHTS INTO SPECIATION -

3.1 Abstract

Drought is one of the most injuries and most frequently encountered natural stresses, and can be magnified in urban and suburban environments. The objective of this study was to evaluate water use characteristics and morphology of seven Cercis L. taxa of different origins, including several measurements of root system water use efficiency. Plants were grown in the greenhouse and sixty days after germination, water use per plant was determined gravimetrically, three times over 24 h. Height, caliper, number of nodes on the main shoot axis and leaf area were measured. Roots were scanned and analyzed with

WinRhizo software to determine root length, root surface area and root diameter. Dry weight of leaves, shoots and roots was obtained. A principal component analysis showed great variances in shoot and root morphology as well as water use characteristics among taxa; it showed a trend towards decreasing water use per unit plant mass with increasing plant size. Cercis chinensis showed a relative high water use per seedling per day, but had the lowest water use per day per cm height, per cm2 leaf area, per g leaf dry weight and per cm root length. Cercis canadensis (OH) used the least amount of water per seedling per day, and had a highly efficient root system based on its absorbance of significantly more water per cm2 root surface area than other taxa. No consistent pattern of characteristics occurred for mesic- or xeric-adapted taxa. Each taxon used a unique set of characteristics to adapt to its original environment.

Taxa used: C. canadensis (OH, OK), C. canadensis var. texensis, C. occidentalis, C. chinensis, C. griffithii, C. siliquastrum

Key words: Water use, root system efficiency, plant morphology

3.2 Introduction

Water deficit limits plant distribution in many geographic areas (Whitlow et al. 1992) and is one of the most important factors affecting plant growth and productivity (Taiz and

Zeiger 2002). Quizenberry (1982) defined drought stress as any period during which plant and/or soil water deficits affect the growth and development of plants. Urban and suburban environments magnify stresses that are common within most landscapes, with drought being the most frequent and injurious stress encountered, since it affects nearly all plant physiological processes (Kramer 1987, Cregg 2004). The selection of trees for urban and suburban environments is often based on their aesthetic contribution to the landscape, with little consideration of their adaptation to the site. Irrigation can mitigate the impact of drought stress but with water restrictions becoming more common throughout the US, increasing water prices, limited availably and ground water pollution; it is becoming less of an option (Schuch and Burger 1987, Knox 1989).

Plants have developed many anatomical, morphological and physiological characteristics to resist drought stress (Flint 1985). Drought resistance, the ability to withstand periods of dryness, includes the avoidance of plant water deficits (drought avoidance) and the ability to tolerate plant water deficits (drought tolerance) (Larcher 2003). Plants can respond to drought by acclimation, which includes the accumulation of solutes to adjust osmotic potential, changes in cell wall elasticity and often morphological changes

(Pugnaire et al. 1999). Plants can also respond by adjusting, within genetic limits, patterns of biomass allocation, anatomical modification and physiological mechanisms including a reduction in growth to balance resource acquisition or even die back. Many of the traits that control plant water status are constitutive and not adaptive (Blum 1996). 77

Roots are the only apparatus for meeting the transpiration demand of a plant and are a key component of plant adaptation to xeric environments (Blum 2002). Allocation of proportionally more dry weight to the roots than the above ground plant is common in xeric-adapted plants. Plants with deep, wide spreading and highly branched root systems maximize water uptake from the soil better than plants lacking these adaptations (Graves

1996). Root-shoot ratio quantifies the biomass allocation to above or below-ground structures for the capturing of light and soil resources (Tilman 1988). However, an increase in root dry weight at the expense of shoot dry weight, without an actual increase in water absorbing capacity (through an increase in percent root surface area or in efficiency) is unlikely to benefit drought resistance (Hinckley et al. 1981). Therefore, the ratio of root surface area to leaf surface area and the associated organ efficiency of water uptake and use are important since they determine the ratio of transpiration-to-absorption potential (Hinckley et al 1981). Some tree species develop extensive root systems as seedlings, prior to shoot growth and others allocate photosynthetic energy preferably to the root system as a response to stress (Graves 1996). The shoot-root ratio is known to decrease under drought conditions but these changes are mainly due to a proportionally greater reduction in shoot, than root growth (Graves 1996, Blum 2005).

St. Hiliare and Graves (2001) suggested selecting plants from populations indigenous to xeric environments as a strategy for identifying ornamentals with superior drought resistance. In contrast, Drunasky and Struve (2005) suggested selecting plants adapted to shallow-sandy soils as an alternative strategy and that drought avoidance associated with a deep root system may be effective in restricted root zones associated with urban

planting sites. This study examines differences in water use and root morphology of

Cercis taxa from North American and worldwide sources.

The genus Cercis is comprised of currently 10 recognized species, native to the warm north-temperate zones of Eurasia and North America (Fritsch et al. 2009). Cercis are small to medium sized trees or shrubs, cultivated widely as ornamental. In North

America, C. canadensis L. is a valuable landscape tree and produced commercially as a nursery crop (Nardini et al. 2003). Cercis canadensis is native to the United States and one of the most widely distributed species, ranging as far west as southeastern Nebraska, western Oklahoma and Texas, and southward into east-central Mexico and west-central

Florida (Donselman and Flint 1982, Clark and Bachtell 1992). Three varieties have traditionally been recognized in the United States: C. canadensis var. canadensis, which is widespread in the Eastern United States, C. canadensis var. texensis L. (S.Watson) M.

Hopkins and C. canadensis var. mexicana L. (Rose) M. Hopkins. C. canadensis var. texensis is found in east-central Texas and adjacent Oklahoma (Hopkins 1942) and is considered highly drought resistant. Cercis occidentalis is native to California, Arizona,

Utah (Davies et al. 2002, National Resources Conservation Center 2010), Nevada

(Hopkins 1942) and Oregon (Raulston 1990). It is considered drought tolerant and adapted to Mediterranean-like summers (Raulston 1990). Cercis siliquastrum L. is commercially important in its natural range throughout the Mediterranean region and southern Europe, from France to Turkey and Afghanistan (Robertson 1976, Rechinger

1986, Davis et al. 2002) but is so widely naturalized that it is difficult to determine its origin. Cercis siliquastrum is well adapted to semi-arid conditions and can withstand hot dry summers provided that soil moisture is adequate in winter and spring. Cercis 79

chinensis L. is native and widely distributed in China. It is a temperate zone tree that occurs in hilly districts but is also found in the open country and the margins of woodlands. Cercis griffithii L. is native to Afghanistan (Raulston 1968) and described to be very similar to and by some considered an ecotype of C. siliquastrum.

In an effort to determine if physiological and morphological differences exist among seven Cercis taxa, seedlings were grown in containers under non-limiting substrate moisture conditions.

3.3 Material and methods

This study used three seed sources of the Cercis canadensis L.: [Ohio (collected at

Chadwick Arboretum, The Ohio State University, Columbus, OH), Pennsylvania

(060509), Oklahoma (060047)], one seed source of C. canadensis var. texensis (collected in Dallas, TX) and four species of worldwide origin (C. chinensis (050748), China; C. griffithii (010370) and C. siliquastrum (070453). All seeds except the Ohio source were obtained from Sheffield’s Seeds Company, Inc. (Locke, NY) (seed lot numbers are given in parenthesis).

In December 2008, seeds were scarified with hot water. After soaking in water over night, the seeds were placed in plastic bags in a refrigerator at 4C. In March 2009 seeds were planted in black plastic containers (13 cm square x 15 cm deep, 1.95 L, Classic 250,

Nursery Supplies, Chambersburg, PA) into a mixture of 2:8 (by vol.) of Metro Mix 852

(Sun Gro Horticulture, Bellevue, WA) to Turface Athletic MVP (Profile Products LLC,

Buffalo Grove, IL). The experiment employed a randomized complete block design with nine single plant replications per taxa. The germination date was noted for each plant. A

Watchdog 400 data logger (Spectrum Technologies, Inc., Plainfield, Illinois) was used to record the environmental conditions (temperature, relative humidity and photosynthetic active radiation (PAR) at canopy height). Three consecutive 24 h whole plant water use measurement were completed 60 d after germination. The substrate was saturated and allowed to drain for one hour to reach container capacity and then weighted (w1). After a period of 24 h, the container weight was determined again (w2). The difference between w1 and w2 equaled the water used during the measurement period. Plants were then re- watered, allowed to drain for 1 h, and weighed which began the measurement period.

After height, caliper and number of leaves were determined; plants were destructively harvested and separated into roots, shoot and leaves. The leaf area was determined with a

LI-3100 leaf area meter (LI-COR, Lincoln, NE). The substrate was completely cleaned from the roots and the roots were then scanned color using a HP Scanjet 4850 (resolution

300 dpi). The scanned images were analyzed using root scanning software (WinRhizo

Pro 2007 d, Regents Instruments Inc., Québec), to determine total root length (cm), total root surface area (cm2), average root diameter (mm), total root length per cubic cm of media (cm/cm3) and total root volume (cm3) for each plant. Water use was averaged per plant and day. Calculated values included: water use per seedling per day, per cm height per day, per cm2 leaf area per day, per g leaf dry weight per day, per g root dry weight per day, per cm root length per day, per cm2 root surface area per day, per mm root diameter per and per cm3 root volume per day. Roots, shoots and leaves were dried at 85 C for

72 h and then weighed.

Statistics

Data was subjected to an analysis of variance using the generalized linear model (PROC

GLM) of SAS 9.2 (SAS Institute, Cary, NC). When significant species differences were found, means were separated by using the Waller-Duncan test at P< 0.05. The variables were also subjected to a principal component analysis (PCA) based on a correlation matrix using PROC FACTOR of SAS 9.2.

3.4 Results

Principal component analysis

Three components accounted for 77% of the cumulative total variance in the principal component analysis (Table 3.1). The first principal component (PC1) accounted for 52% of the total variance and represented plant growth and water use characteristics. Plant growth characteristics were positively correlated with PC1. The highest correlations with

PC1were leaf dry weight, total dry weight and root surface area. Most water use characteristics were negatively correlated with the first component (water use cm-2 leaf area per day, water use per g leaf dry weight per day, water use per g root dry weight per day, water use per cm root length per day and water use cm-2 root surface area per day).

The first principal component showed a trend towards decreasing water use per unit plant mass with increasing plant size. For instance, tall plants had lower water use per cm2 leaf area per day than small plants.

The second principal component (PC2) accounted for 16% of the total variance and displayed the relative investment by the plant into above and below ground biomass

(Table 3.1). Root growth variables (root dry weight, root length, root surface area, root surface area-to-leaf area, root length-to-leaf area) were positively correlated with PC2, while most other plant growth variables were negatively correlated.

The third component (PC3) accounted for 9% of the total variance (Table 3.1). Water use per seedling per day and water use per cm height per day were highly and positively correlated with PC3.

Variable PC 1 PC 2 PC 3 Height (cm) 0.86 -0.29 0.15 Caliper (mm) 0.86 -0.03 0.26 No. of leaves 0.76 -0.08 0.27 Leaf area (cm2) 0.87 -0.33 0.03 Dry weight (g) Leaf 0.94 -0.17 0.11 Shoot 0.70 0.01 0.15 Root 0.78 0.43 0.08 Total 0.96 0.02 0.12 Root length (cm) 0.86 0.28 -0.02 Root surface area (cm2) 0.90 0.35 0.07 Specific root length (cm/g) 0.84 0.36 -0.17 Shoot root ratio 0.48 -0.66 0.12 Root surface area over leaf area -0.18 0.92 0.10 Root length over leaf area -0.18 0.87 -0.01 Water use (g) per day per seedling 0.42 0.08 0.75 per cm height -0.13 0.29 0.61 per cm2 leaf area -0.80 0.31 0.38 per g leaf dry weight -0.76 0.22 0.32 per g root dry weight -0.60 -0.42 0.49 per cm root length -0.75 -0.29 0.44 per cm2 root surface area -0.61 -0.24 -0.02 Cumulative percent of total variation explained 52 68 77

Table 3.1 Rotated factor pattern for principal component analysis of seven Cercis L. taxa. Loadings above 0.60 are marked in bold.

Cercis canadensis (OH), C. canadensis (OK), C. can. var. texensis and C. chinensis clustered together on PC2 (Fig. 3.1), associated by high shoot-root ratios and low root surface area to leaf area and root length to leaf area (Table 3.2). Cercis chinensis were separated from these other taxa on PC1. Cercis chinensis was the largest of the taxa with regard to height, caliper, number of leaves, leaf area, leaf- and shoot dry weight, total dry weight (Table 3.2) and root length (Table 3.3). Although C. chinensis showed a relative

high water per seedling per day, these plants had the lowest water use per day per cm height, per cm2 leaf area, per gram leaf dry weight and per cm root length (Table 3.4).

0.8 B

0.6 G E 0.4 D 0.2

0.0

PC3 F -0.2 C 1.2 -0.4 1.0 0.8 -0.6 0.6 0.4 0.2 -0.8 A 0.0 PC1 1.0 -0.2 0.5 -0.4 0.0 -0.6 -0.5 -0.8 PC2 -1.0

Fig. 3.1 Plotted values of the first three principal components of Cercis L. taxa.

A= C. canadensis (OH), B= C. occidentalis, C= C. canadensis (OK), D= C. canadensis var. texensis, E= C. chinensis, F= C. griffithii, G= C. siliquastrum.

Dry weight No. of Taxa Height Caliper Leaf area Leaf Shoot Root Total leaves cm mm cm2 g g g g C. canadensis (OH) 8.2y dz 1.2 c 5 c 84 bc 0.4 bc 0.09 0.23 b 0.6 b C. occidentalis 10.7 bcd 1.7 ab 7 ab 72 c 0.4 bc 0.08 0.30 ab 0.8 ab C. canadensis (OK) 11.3 bc 1.5 bc 6 b 93 cb 0.5 bc 0.07 0.23 b 0.7 ab C. can. var. texensis 12.6 b 1.5 bc 6 b 115 b 0.5 ab 0.07 0.26 b 0.9 ab C. chinensis 16.6 a 1.9 a 8 a 156 a 0.7 a 0.11 0.27 ab 1.0 a C. griffithii 8.0 d 1.3 c 5 c 67 c 0.3 c 0.03 0.23 b 0.6 b

86 C. siliquastrum 9.8 bcd 1.6 ab 7 b 92 bc 0.4 bc 0.09 0.37 a 0.9 ab

P > F <.00001 0.0003 <0.0001 0.0005 0.0074 0.2776 0.0332 0.0635

Table 3.2 Plant growth characteristics of seven Cercis taxa L. of different origin.

y Each value is the mean of nine plants per taxon.

z Means within a column followed by different letters are significantly different from each other at α 0.0 using the Waller- Duncan test of significance

Taxa Root length Root surface Specific root Root Shoot- Root surface Root area length diameter root ratio area/leaf area length/leaf area cm cm2 cm g-1 mm cm2 cm-2 cm cm-2 C. canadensis (OH) 1006y cdz 176 b 5.1 0.57 c 1.9 cd 2.3 cd 12.8 b C. occidentalis 838 d 204 b 4.5 0.81 a 1.6 d 2.8 bc 11.6 b C. canadensis (OK) 1098 bcd 198 b 5.7 0.58 c 2.5 ab 2.1 d 11.9 b C. can. var. texensis 1355 abc 233 ab 5.2 0.54 c 2.3 bc 2.3 cd 13.1 b C. chinensis 1805 a 297 a 7.4 0.54 c 2.9 a 1.9 d 12.0 b C. griffithii 1164 bcd 197 b 5.0 0.55 c 1.4 d 3.1 ab 18.4 a 87 C. siliquastrum 1579 ab 305 a 8.3 0.63 b 1.4 d 3.5 a 17.8 a

P > F 0.0015 0.0107 0.3385 <0.0001 <0.0001 <0.0001 0.0007

Table 3.3 Root growth characteristics and shoot-to-root relations of seven Cercis taxa of different origin.

y Each value is the mean of 9 plants per taxa.

z Means within a column followed by different letters are significantly different from each other at α 0.0 using the Waller- Duncan test of significance

Water use per day per leaf dry root dry cm cm2 root Taxa seedling cm height cm2 leaf area weight weight root length surface area g g g g g g g

C. canadensis (OH) 51.4y d z 7.1 abc 0.8 ab 205.7 ab 248.7 0.06 b 1.17 a

C. occidentalis 65.2 abc 6.8 abcd 1.1 a 180.5 ab 262.3 0.10 a 0.40 b

C. canadensis (OK) 55.8 cd 5.2 cd 0.7 ab 141.9 b 306.1 0.06 b 0.35 b

C. can. var. texensis 69.4 ab 5.9 bcd 0.7 ab 145.7 b 276.1 0.05 b 0.32 b

88 C. chinensis 69.9 ab 4.5 d 0.5 b 112.7 b 279.3 0.04 b 0.25 b

C. griffithii 60.7 abc 8.4 a 1.1 a 293.0 a 299.6 0.06 b 0.38 b

C. siliquastrum 71.4 a 7.8 ab 0.8 ab 183.2 ab 198.8 0.05 b 0.24 b

0.0005 0.0062 0.0182 0.0256 0.4738 0.0246 <0.0001

Table 3.4 Water use characteristics of seven Cercis of different origin.

y Each value is the mean of 9 plants per taxon. z Means within a column followed by different letters are significantly different from each other at α 0.0 using the Waller- Duncan test of significance 88

Cercis canadensis (OK) and C. canadensis var. texensis showed no significant differences in plant growth characteristics or water use characteristics which loaded on

PC1, but did show differences in loadings on PC3 due to a significantly higher water use per seedling per day of C. canadensis. var. texensis compared to C. canadensis (OK). The separation of C. canadensis (OH) on the PC1 axis was attributed to its high water use relative to its plant size. Cercis canadensis (OH) were the smallest plants with regard to height, number of leaves, leaf area, leaf dry weight, root dry weight (Table 3.2) and root surface area (Table 3.3), but used the most water per day per cm2 leaf area, per gram leaf dry weight, per cm root length and especially per cm2 root surface area. These statistics where almost triple those of C. occidentalis, which had the second highest water use.

Cercis griffithii and C. siliquastrum were similar in terms of a relatively low shoot-root ratio and high root surface area-to-leaf area and root length-to-leaf area ratio. This placed both species opposite to C. canadensis (OH), C. canadensis (OK), C. canadensis var. texensis and C. chinensis on PC2. However, differences between these species were in their loadings on PC1; C. siliquastrum seedlings had a significantly greater caliper, higher number of leaves, greater root dry weight, a much larger root surface area and larger root diameter than C. griffithii seedlings. Differences in water use between these two species did not contribute to differences in their loadings on the PC1 axis. Cercis siliquastrum was similar to C. canadensis var. texensis in most plant growth and water use characteristics as indicated by their similar loadings on PC1. However, both species differed significantly in their loadings of PC2, due to C. siliquastrum having lower shoot- root ratio and higher root surface-to-leaf area and root leaf to leaf area ratios. Cercis occidentalis was intermediate between the other taxa. In terms of plant growth and water

use characteristics which loaded positively on PC1, C. occidentalis showed strong similarity to C. canadensis (OK). Only root diameter was significantly greater in C. occidentalis than C. canadensis (OK). However, a relatively high water use per seedling per day and an additional high water use per cm height per day, separated C. occidentalis on PC3.

The taxa showed significant differences in all plant growth characteristics (Table 3.2) with the exception of shoot- and total plant-dry weight. Cercis chinensis seedlings were the largest plants while C. canadensis (OH) and C. griffithii were the smallest taxa, especially with regard to height and total plant dry weight. Root characteristics and root- to-shoot relations (Table 3.3) were significantly different for the taxa with exception of specific root length. Cercis chinensis had the highest root length; however, C. siliquastrum had the highest root surface area. Cercis occidentalis had the shortest root length but the greatest root diameter. Cercis siliquastrum had the highest root surface area and root-surface-area to leaf-area ratio, but had also the lowest shoot-to-root ratio.

All water use characteristics except water use per cm root length per day were significant

(Table 3.4). Cercis siliquastrum used the most water per seedling per day. Cercis chinensis showed a similar high water use per seedling per day as C. siliquastrum, but used the least water per day per cm height or per cm2 leaf area or per leaf dry weight or per cm root length. Cercis canadensis (OH) on the other hand, used the least amount of water per seedling per day, but absorbed significantly more water per cm2 root surface area than any other taxa, indicating a highly efficient root system.

3.5 Discussion

The principal component analysis suggests clinal variation among the North American taxa, C. canadensis (OH), C. occidentalis, C. canadensis (OK) and C. canadensis var. texensis, based on loadings on PC1 (plant growth and water use per unit plant mass/area).

No clustering was evident with regard to the other taxa under the conditions of this experiment (juvenile plants and non-limiting soil moisture conditions).

No consistent pattern of plant growth or water use characteristics employed by mesic- or xeric-adapted taxa was found. Even among the xeric-adapted taxa, each taxon expressed a unique set of characteristics. As in Chapter 2, these morphologies were expressed in juvenile seedlings grown under non-limiting soil moisture conditions.

Deep, wide-spreading and highly branched root systems maximizes the uptake of water from the soil and allows the plant to avoid drought injury for a longer period of time

(Kramer 1983, Graves 1996) than plants with shallow, coarse root systems. In some species, preferential allocation of photosynthate to the root system over the shoot, occurs regardless of the soil moisture conditions, while other species develop an extensive root system only in response to drought (Graves 1996). One of the major traits in determining a plants ability to acquire soil moisture is maximum root depth. Maximum root depth depends on root length, a constitutive trait, and the plant’s interaction with the environment.

Root length varied widely within the Cercis taxa in this study. Cercis occidentalis, considered adapted to a xeric environment, had the lowest root length; while C. chinensis

(considered mesic adapted) had a root system more as twice as long. Cercis occidentalis 91

also had the lowest specific root length (SRL). Low SRL results from shorter root length

(smaller root surface area) per unit carbon invested. Some studies have found that fast- growing species have a higher SRL than species from more xeric environments (Ryser and Eek 2000, Craine et al. 2001, Nicotra et al. 2002, Comas and Eissenstat 2004), while other studies have shown that species from xeric environments have higher SRL than species from mesic environments (Poot and Lambers 2003, Tjoelker et al. 2005). In this study there was no difference in SRL among the taxa, however, C. occidentalis had the lowest SRL, while C. siliquastrum, another xeric adapted species had the highest (twice as high as C. occidentalis). The lack of significant difference in SRL was likely due to a small sample size.

A low shoot-root ratio indicates a large root system and a high water absorbing capacity

(Graves 1996). In this study C. occidentalis, C. griffithii and C. siliquastrum had the lowest shoot-root ratio; C. canadensis (OK) and C. chinensis the highest. The nursery industry favors species that rapidly develop a vigorous canopy, not species that allocate resources to the root rather than the shoot (Graves 1996). However, if higher drought resistance of slower growing taxa can be documented, then slower growing taxa might be favored over faster growing but less drought resistant taxa.

Cercis canadensis var. texensis and C. chinensis were the tallest plants with the greatest leaf area and total plant dry weight. Both taxa had high rates of water use per seedling per day. Cercis siliquastrum on the other hand was significantly shorter, and had less leaf area but comparable total plant dry weight and the highest water use per seedling per taxa. Different life strategies are visible here. All three taxa have high rates of water per

seedling per day, but while C. canadensis var. texensis and C. chinensis had high shoot- root ratios, and relatively low ‘root surface-to-leaf area ratio’ or ‘root length-to-leaf area ratio’ to support this demand, C. siliquastrum was able to meet its water demand because of its much smaller size and significantly greater root surface and ‘root length-to-leaf area ratio’.

Cercis siliquastrum displayed the typical growth habit of drought resistant species, which, even when there is ample water available often grow slow (Passioura 1983).

Cercis canadensis had the lowest water use per seedling and the lowest root surface area, but the most efficient root system in terms of water use per cm2 root surface area.

Chapter 4

PHOTOSYNTHESIS, CHLOROPHYLL FLUORESCENCE AND WATER USE OF SEVEN CERCIS L. TAXA DURING DROUGHT STRESS AND RECOVERY

4.1 Abstract

Drought is one of the most frequently encountered stresses by plants. Plants have developed morphological, anatomical and physiological adaptations to resistance drought. The objective of this study was to evaluate morphological and physiological differences in response to water deficit. Seedlings of seven Cercis taxa from different origin were submitted to a water deficit and recovery cycle under greenhouse conditions.

Net photosynthesis, stomatal conductance, intrinsic water use efficiency and chlorophyll fluorescence, as well as plant growth and water use were measured. Paraheliotropism was observed in all taxa at varying degrees. All taxa reduced net photosynthesis and stomatal conductance shortly after exposure to water deficit. However, C. occidentalis and C. siliquastrum had high initial values for net photosynthesis and stomatal conductance and showed a relatively lower decline in both values than other taxa. Chlorophyll fluorescence declined in some taxa during the period of water deficit, while other taxa where unaffected. However, during the recovery period affected taxa showed increasing values for net photosynthesis, stomatal conductance and chlorophyll fluorescence. I concluded that taxa in our study are drought-resistant and employ several drought resistant mechanisms; the ability of plants to withstand drought stress without altering the function of the PSII system; the capacity of plants to quickly restore the damage to the photochemical apparatus and paraheliotropism. Cercis taxa also displayed large within taxa variation. Cercis canadensis var. texensis would be an excellent candidate for inducing drought resistance into a breeding program based on its relative drought resistance, growth habit and rapid growth rate.

Taxa used: C. canadensis (PA, OH, PA), C. canadensis var. texensis, C. occidentalis, C. siliquastrum, C. chinensis.

Key words: Photosynthesis, stomatal conductance, chlorophyll fluorescence, water use efficiency, drought, recovery

4.2 Introduction

The genus Cercis comprises small to medium sized trees or shrubs. Ten species are recognized, originating from the warm north-temperate zones of North America and

Eurasia (Fritsch et al. 2009). Cercis canadensis is the most important species in the

United States, being a valuable landscape tree that is commercially produced as a nursery crop (Nardini et al. 2003). Three varieties of C. canadensis are recognized: C. canadensis var. canadensis which is widespread in the Eastern United States, and C. canadensis var. texensis (S. Watson) M. Hopkins and C. canadensis var. mexicana (Rose) M. Hopkins, which are native to xeric environments in the United States. Cercis occidentalis, the western redbud, is the second most common North American species, native to

California, Arizona and Utah (Hopkins 1942, Isley 1975, Correll and Hohnston 1979,

Turner et al. 2003). Cercis siliquastrum is native to the Mediterranean region and southern Europe (Robertson 1976, Rechinger 1986, Davis et al. 2002) and C. griffithii is native to Afghanistan (Raulston 1968). Five Cercis species, including C. chinensis are indigenous to China (Woodward and Williams 1987, Pu Zou et al. 2008).

Due to its small size (up to 12 m tall), C. canadensis is often used in residential gardens in the United States (Clark and Bachtell 1992) and other restricted spaces such as parking lots, buffer strips and as a street tree. However, the usage of C. canadensis for this purpose is limited since the lifetime of Cercis in urban settings is only about 15-20 years.

However, if the tree is not subjected to stresses such as drought, which are common in urban environments; it can be an attractive tree for decades (Clark and Bachtell 1992).

Drought is one of the most frequently encountered stresses of plants in the urban and suburban landscape. Drought is a multi-dimensional stress that affects virtually all 97

physiological processes directly or indirectly (Kramer 1987, Cregg 2004) and has impact on photosynthesis, respiration, protein synthesis and carbohydrate metabolism (Pallardy

2008). Plant growth is especially sensitive to water deficits and is reduced by even mild stress (Jones 1992, Kozlowski et al. 1991).

Stomatal closure is among the earliest plant responses to water deficit and the most important factor in controlling carbon fixation (Yordonov et al. 2000). A reduction in photosynthesis may be due to stomatal limitations (stomatal closure and the increased resistance to diffusion of CO2). Additionally non-stomatal limitations to carbon fixation can occur with damage to the reaction centers of photosynthesis I and II (inhibition of the photosynthetic apparatus (Yordanov et al. 2000, Pallardy 2008)). Changes in the photochemical efficiency of drought-stressed plants may be assessed by analysis of chlorophyll a fluorescence associated with PSII.

Trees for urban and suburban landscapes are most often selected for their aesthetic value, with little consideration of their adaption to the given site. While irrigation can mitigate some potential problems, with water restriction becoming more common throughout the

United States, increasing water prices and limited availability and pollution of ground water, it is becoming less of an option (Schuch and Burger 1987, Knox 1989). The best strategy to improve survival, growth and health of trees in urban and suburban landscapes may be to select trees with improved drought resistance and reduced water use. Further, although morphological, molecular, and biochemical differences exist among the North

American Cercis taxa, information regarding physiological differences among Cercis taxa is lacking. The objective of this study was to investigate water use, gas exchange,

chlorophyll fluorescence as well as plant growth in seedlings of Cercis taxa from different origins when submitted to a water deficit and recovery cycle and to determine the drought resistance mechanisms and the relative drought resistance among the taxa.

4.3 Material and methods

Seven sources of Cercis were used in this experiment; the species C. chinensis (050748),

C. occidentalis (060423) and C. siliquastrum (070453), as well as three seed sources of

C. canadensis (collected in Pennsylvania (060509), Ohio (collected at Chadwick

Arboretum, The Ohio State University, Columbus, OH) and Oklahoma (060470), and one source of C. canadenis var. texensis (collected in TX). All seeds, expect the Ohio source and C. canadensis var. texensis, were obtained from Sheffield’s Seed Company, Inc.

(Locke, NY), (seed lot numbers are given in parenthesis).

In October 2008 the seeds were scarified with hot water and stored in a cooler at 4°C until March 2009 when the seeds were planted into flats filled with SunGrow Metro Mix

852 (Sun Gro Horticulture Canada Ltd., Bellevue, WA) to germinate. In April 2009 they were transplanted into plastic containers (13 cm square x 15 cm deep, 1.97 L, Classic

250, Nursery Supplies, Chambersburg, PA) using SunGrow Metro Mix 852 substrate.

Plants were fertilized at 0 ppm N once a week with a water soluble fertilizer (Peter’s

Excel 15-5-15 Cal Mg, The Scotts Company LLC, Marysville, OH) and at 100 mg N per week from May on. In June the plants were transplanted in # 1 containers (17.8 cm H x

19.7 cm D, 3.8 L, C400, Nursery Supplies Inc., Chambersburg, PA) and spaced (0.75 cm on center) on two benches, in a greenhouse for establishment. The greenhouse temperature was set at 24°C and cooled with fan and pad cooling; seedlings were grown under natural photoperiods.

The experiment employed a split plot design with seven Cercis taxa and two water stress treatments, water deficit or a well-watered control, with six single plant replications per

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treatment combination. The experimental plot on each bench was surrounded by one row of guard plants to ensure homogeneous conditions. A Watchdog 400 data logger

(Spectrum Technologies, Inc., Plainfield, IL) was used to record temperature, relative humidity and photosynthetic active radiation.

Plants were irrigated daily by hand until substrate saturation before exposure to a water deficit and recovery cycle. Beginning July 28th 2009 plants were exposed water deficit by withholding water from half the plants within a taxon, whereas control plants were watered daily throughout the study. The period of water deficit was terminated by watering the non-watered plants daily from August 8th to the 23rd 2009, when the experiment was terminated. Subsamples of six plants per taxa were harvested on July 28th

2009 (initial); at the termination of water deficit and on August 23rd 2009 six additional plants per taxa and treatment were harvested. (See later section for description of plant harvests).

Substrate samples

Substrate samples from all plants were also taken just before termination of water deficit by collecting 30 g of substrate from each harvested plant. The samples were collected by removing the plants from their container and teasing substrate vertically from the root ball. The samples were weighed (w1), dried for 24 h at 105°C and then re-weighed (w2).

The moisture content of the substrate was calculated as: (w2/w1) * 100

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Substrate moisture release curve

To describe the water storage in the substrate and its availability to plants, a substrate moisture release curve was established. The substrate was spread out on plastic sheets on a greenhouse bench to air dry for 5 d. Then, 30 g each of the substrate was taken and wetted by incrementally adding 1.5 g of water to each of 3 subsamples. The wetted samples then were sealed in an airtight container and allowed to equilibrate for 24 h. The substrate moisture curve was developed using a Decagon WP4 Dewpoint potentiometer

(Decagon Devices, Inc., Pullman, WA). Three samples of each water content were measured and then their mass immediately determined. The samples were oven-dried for

24 h at 105°C. After removal from the oven the samples were placed in a dessicator for about 10 minutes to cool. The cooled samples were weighed and the water content calculated as follows:

Sample wet (g) – sample dry (g) = substrate water content (g/g).

The results were then plotted against the water potential measured by the dew point potentiometer.

Water use

Whole plant water use was measured gravimetrically for each plant. The substrate was saturated and allowed to drain for 1 h to reach container capacity and then weighed (w1).

After 24 h the container was reweighed (w2). The difference between w1 and w2 was the

24 h water use (evapotranspiration).

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Morphology

At beginning, at time of drought termination and at the end of the experiment, plant height and caliper was measured. The nodes on the main stem and the number of basal nodes without leaves (an indicator of leaf abscission) were counted. All plants were destructively harvested at the end of the experiment. Due to large differences in plant size among the taxa at the beginning of the experiment, plant growth data will be reported as changes during the period of water deficit and at the end of the recovery period separately.

Dry weight and LAR, NAR, RGR

Initially, six plants per taxa were destructively harvested and used as initial values for the calculation of dry weight change and leaf area ratio (LAR, cm2 g-1), net assimilation rate

(NAR, mg cm-2 d-1) and relative growth rate (RGR mg mg-1 d-1) during the subsequent experimental period. Leaf area was determined by using a LiCor 3100 leaf area meter

(LiCor, Lincoln, NE). Dry weight of leaves, shoots and roots were determined by separating the plant parts, washing the substrate of the roots and drying the plant parts for

48 h at 57 oC. Changes in dry weights were calculated from the initiation of the experiment to the time of maximum water deficit and to the end of the experiment as well as for the entire duration of the experiment. The three sets of plants needed for these calculation, were sorted by increasing dry weight per harvest and paired to determine changes in dry weight, and calculation of RGR, NAR and LAR. Therefore, the plant with the least dry weight at the beginning of the experiment was paired with the plant with least dry weight at time of maximum water deficit and the plant with the least dry weight

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at the end of the experiment. The NAR and LAR were calculated according to Harper

(1977): NAR = [(W2-W1)/(t2-t1)x(loge L2-loge L1)/(L2-L1)] and LAR = [(L2-L1)/(W2-W1) x (loge W2-loge W1)/(loge L2-loge L1)]. The RGR was calculated as the product of NAR and LAR.

Gas exchange and chlorophyll fluorescence

Gas exchange measurements were taken using a LiCor 6400 (LiCor Biosciences,

Lincoln, NE) with a chlorophyll fluorescence chamber as light source and using the following settings: CO2-S: 370 ppm, flow rate: 400, block temperature: 28C, PAR: 2000

μmol m-2 s-1 (with 10% blue) and RH at 40-42%. One randomly selected fully matured attached leaf per plant was measured. During the period of water deficit gas exchange and chlorophyll fluorescence measurements were performed every second day, and during the recovery period every third day. Chlorophyll fluorescence measurements were performed daily on the same plants as the gas exchange measurements. Three randomly selected leaves per plant were measured using a Chlorophyll Fluorometer (OptiSci OS-

30p, Opti-Sciences, Inc., Hudson, NH).

The taxa displayed great differences in initial values for photosynthesis and stomatal conductance. Therefore, these values are displayed as the percentage of change from the beginning of the experiment to the time of maximum water deficit and to the end of the recovery period.

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Observations

At time of maximum water deficit observations were made on leaf orientation and wilting pattern, and senescence/abscission of the main shoot terminal. Special attention was paid to paraheliotropic movements of the leaves.

Statistics

The experiment was set up and analyzed as split-plot design with water-stress treatment as main-treatment and species as the sub-treatment. The main effect was replicated six times; six single plant replications per treatment combination were used for a total of 84 plants. Data were subjected to analysis of variance using the generalized linear model

(PROC GLM) of SAS (SAS Institute, Cary, NC) as a fixed effect model. When significant treatment effects occurred, means were separated by the Waller-Duncan mean separation test at P< 0.05 level of significance; for physiological and dry weight data the

Tukey’s mean separation was employed at P< 0.05 level of significance.

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4.4 Results

Soil moisture

After 9 d of withholding water substrate moisture of non-watered plants reached -8.55 to

-185.28 MPa, while the control plants had substrate moistures of -0.69 to -0.87 MPa; indicating that all non-watered plants were exposed to low soil water potentials (Fig. 4.1).

200

y = 0.0087x-3.479 R² = 0.9419

150

MPa) -

100

Water potential ( potentialWater 50

0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Water content (g/g)

Fig. 4.1 Substrate moisture release curve for SunGrow MetroMix (●) and substrate moisture content (▲) of non-watered plants and watered control plants (■) of Cercis after 9 d of withholding water.

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Water use and intrinsic water use efficiency

Control plants and non-watered plants showed no difference in water use for the first 24 h after withholding water (Fig. 4.2 A-G). However, for all but one taxon (C. occidentalis) non-watered plants used less water than control plants after 7-8 d of withholding water

(Table 4.1). All taxa showed significant differences in water use on the first 2 d after re- watering, with control plants using more water than non-watered plants (Table 4.1).

Cercis canadensis (PA), C. occidentalis, C. canadensis var. texensis, C. chinensis and C. siliquastrum displayed differences in water use between control and non-watered plants for only a few days and no significant differences after 7 d of recovery period. Non- watered plants of C. canadensis (OH) and C. canadensis (OK) on the other hand had lower water use on most days of the water deficit and recovery cycle. However, on the last day of the recovery cycle, no significant differences in water use were found between watered and non-watered plants of any taxa.

Averaged over all taxa, control plants used 79% more water at maximum water deficit than at the beginning of the experiment, while non-watered plants used 35% less (Fig. 4.2

A-G). This difference in water use, between watered and non-watered plants, decreased towards the end of the recovery period with control plants using 21%, and non-watered plants using 6% more water than at the initiation of the experiment. Over the whole course of the experiment, control plants used 50% more water compared to the beginning of the experiment, while non-watered plants used 14% less.

Intrinsic water use efficiency (WUE) was affected by taxa at all periods of the experiment, at the end of the recovery period and in total. Cercis occidentalis had the

107

lowest intrinsic WUE at each measurement time during the experiment and in total (Fig.

4.2 C). Cercis canadensis (OK) had the highest intrinsic WUE at time of maximum water deficit and in total (Fig. 4.2 D). However, at the end of the recovery period C. canadensis

(PA) had the highest intrinsic WUE (Fig. 4.2 A).

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Days Cercis canadensis Cercis

Drought (PA) (OH) (OK) var. texensis occidentalis chinensis siliquastrum 1 NSz NS NS NS NS NS NS 2 NS NS NS NS NS NS NS 3 NS * NS NS NS * NS 4 NS * NS * NS NS NS 5 NS * NS ** NS NS NS 6 NS * NS NS NS NS NS 7 * * *** *** ** NS ** ** 8 NS * * ** NS NS * Continued

109

Table 4.1 Differences in water use in g between watered control plants and non-watered plants of seven Cercis L. taxa during a water deficit and recovery cycle.

z NS, *, ** and *** indicate no statistical difference at α 0.0 level or significant at α = 0.05, α = 0.01 or α = 0.001 level, respectively.

109

Table 4.1 continued

Recovery 1 *** *** *** *** ** *** *** 2 *** *** *** ** ** *** *** 3 NS ** * * * NS * 4 NS * * NS NS * NS 5 NS * * NS NS NS NS 6 NS NS NS NS NS NS NS 7 NS ** * NS NS NS * 8 NS * ** NS NS NS NS 9 NS NS NS NS NS NS NS 110 10 NS * * NS NS NS NS 11 NS ** * NS NS NS NS 12 NS ** ** NS NS NS NS 13 NS ** * NS NS NS NS 14 NS * * NS NS NS NS 15 NS * NS NS NS NS NS 16 NS ** * NS NS NS NS 17 NS * * NS NS NS NS 18 NS NS NS NS NS NS NS

110

Fig. 4.2 A. Cercis canadensis (PA)

0.6 250

0.5 200

0.4

150 0.3

WUE

WU (g) 100 0.2

50 0.1

0.0 0 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

-1 Fig. 4.2 A-G. Intrinsic water use efficiency (WUE, μmol CO2 mol H2O) and water use (WU, g water per seedling) of seven Cercis taxa exposed to a water deficit and recovery cycle. Black lines represent intrinsic water use efficiency; red lines represent whole plant water use. Solid lines represent the control treatment; dashed lines represent the non- watered treatment. Arrows represent time of re-watering.

Continued

111

Fig. 4.2 continued: B. Cercis canadensis (OH)

0.6 250

0.5 200

0.4 150

0.3

WUE

WU (g) 100 0.2

50 0.1

0.0 0 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

Fig. 4.2 C. Cercis occidentalis

0.6 250

0.5 200

0.4

150 0.3

WUE

WU (g) 100 0.2

50 0.1

0.0 0 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

112

Continued

Fig. 4.2 continued: D. Cercis canadensis (OK)

0.6 250

0.5 200

0.4

150 0.3

WUE

WU (g) 100 0.2

50 0.1

0.0 0 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

Fig. 4.2 E: Cercis canadensis var. texensis

0.6 250

0.5 200

0.4 150

0.3

WUE

WU (g) 100 0.2

50 0.1

0.0 0 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date 113

Continued

Fig. 4.2 continued: F. Cercis chinensis

0.6 250

0.5 200

0.4 150

WUE

WU (g) 0.3 100

0.2 50

0.1 0 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

Fig. 4.2 G: Cercis siliquastrum

0.6 250

0.5 200

0.4

150 0.3

WUE

WU (g) 100 0.2

50 0.1

0.0 0 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

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Initial plant morphological characteristics and dry weight

The taxa used in this experiment showed significant differences for all parameters measured, with exception of the number of leaves on the main shoot axis (Table 4.2 and

4.3). Overall, C. canadensis (PA) had the tallest plants, with the largest caliper, and highest number of nodes on the main shoot axis (Table 4.2). Plants of C. siliquastrum, C. occidentalis and C. chinensis were the shortest. The number of leafless basal nodes on the main shoot axis was greatest in plants of C. occidentalis, while plants of C. canadensis

(OH) had the fewest leafless basal nodes. The largest plants, C. canadensis (PA and OK) and C. canadensis var. texensis had the greatest leaf dry weight and were not significantly different from each other (Table 4.3). Cercis canadensis var. texensis however, displayed lower root dry weight than the two other taxa, resulting in the highest shoot-root ratio.

Cercis canadensis, C. occidentalis and C. siliquastrum had the lowest shoot-root ratio and also the lowest total plant dry weight. Leaf area was largest in plants of C. canadensis (PA) and lowest in C. occidentalis and C. siliquastrum (Table 4.3).

Control plants of the taxa observed maintained their relative growth and dry weight characteristics throughout the duration of the experiment (data not shown). Therefore, only changes in plant growth and dry weight from the initial harvest were reported.

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Taxa Height Caliper Leafless basal nodes Nodes cm mm No. No. C. canadensis (PA) 132.5y az 8.4 a 2.8 b 24.5 a C. canadensis (OH) 108.3 b 7.3 b 1.8 c 21.3 b C. occidentalis 91.4 c 7.0 b 3.9 a 25.3 a C. canadensis (OK) 116.6 b 7.4 b 2.4 bc 24.8 a C. canadensis var. texensis 115.9 b 7.2 b 1.8 c 24.1 ab C. chinensis 91.9 c 7.0 b 2.2 bc 25.8 a C. siliquastrum 83.8 c 7.0 b 2.9 b 24.5 a P > F <0.0001 0.0045 0.0005 0.0374

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Table 4.2 Plant morphological data of seven Cercis L. taxa at start of a water deficit and recovery cycle.

y Each value is the mean of six plants per taxon.

z Means within a column followed by different letters are significantly different from each other at α 0.0 using the Waller- Duncan test of significance.

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Dry weight (g) Shoot-root Leaf area ratio (cm2) Taxa Leaves Shoots Roots Total C. canadensis (PA) 16.0y az 13.7 a 9.1 a 38.8 a 3.5 ab 3277 a C. canadensis (OH) 7.8 b 5.1 c 6.2 a 19.2 c 2.2 b 1641 cd C. occidentalis 7.2 b 8.2 bc 5.4 a 20.8 c 3.0 ab 1254 d C. canadensis (OK) 15.8 a 14.1 a 8.9 a 36.2 ab 3.0 ab 2849 ab C. canadensis var. 16.8 a 13.4 a 6.5 a 36.6 ab 5.3 a 2433 b texensis C. chinensis 12.8 a 9.4 b 7.6 a 29.8 b 3.0 ab 2317 bc C. siliquastrum 6.5 b 6.6 bc 5.9 a 19.0 c 2.3 b 1208 d

117 P > F <0.0001 <0.0001 0.0214 <0.0001 0.0131 <0.0001

Table 4.3 Dry weight of seven Cercis taxa at beginning of a water deficit and recovery cycle.

y Each value is the mean of six plants per treatment combination.

z Means within a column followed by different letters are significantly different from each other at α 0.0 using the Waller- Duncan test of significance.

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Plant growth increase during water deficit and recovery cycle

The increase in height and the number of nodes on the main stem were not significantly affected by water deficit or by taxa during the water deficit and recovery cycle (data not presented). The increase in caliper was significantly reduced during water deficit in non- watered plants compared to control plants (Table 4.4). Control plants of C. canadensis

(OH and OK) and C. siliquastrum increased in caliper by more than twice that of non- watered plants. Differences in caliper increase were observed among the taxa during the recovery period. Cercis canadensis (OH) and C. canadensis var. texensis increased at least three times as much C. occidentalis. Taxa and treatment main effects, but no interaction was observed for increase in caliper during the experiment. Cercis canadensis var. texensis showed a significantly greater increase in caliper than C. occidentalis, C. canadensis (OK) and C. siliquastrum. Non-watered plants increased less in caliper than control plants. The increase in number of leafless basal nodes during the period of water deficit was not affected by treatment (Table 4.4). A significant taxa by treatment interaction was observed during the recovery period for the number of leafless basal nodes, the increase was greater in control plants of C. canadensis (PA) than in previously non-watered plants, while all other taxa showed the opposite or no difference between increase for control and non-watered plants.

118

Caliper (mm) Leafless basal nodes (No.) Species Treatment Water deficit Recovery Total Water deficit Recovery Total C. can. Control 0.52z 0.50 1.02 2.0 3.2 1.2 (PA) Non-watered 0.48 0.50 0.98 0.7 2.0 1.3 Average 0.50 0.5 1.0 1.3 2.6 1.3 C. can. Control 0.60 0.85 1.45 0.3 1.2 0.8 (OH) Non-watered 0.28 0.35 0.63 0.3 4.2 3.8 Average 0.44 0.60 1.04 0.3 2.7 2.3 C. Control 0.32 0.16 0.50 1.2 2.4 1.2 occidentalis Non-watered 0.18 0.20 0.38 0.5 2.8 2.3

119 Average 0.25 0.18 0.44 0.9 2.6 1.8

Continued

Table 4.4 Changes in caliper size and number of leafless basal nodes for Cercis plants from seven taxa exposed to an 8 d period of water deficit and 18 d recovery period.

z Each value is the mean of six plants per treatment combination.

119

Table 4.4 continued

C. can. (OK) Control 0.52 0.38 0.90 0.5 2.0 1.5 Non-watered 0.22 0.42 0.63 3.5 8.8 5.3 Average 0.37 0.40 0.77 2.0 5.4 3.4 C. can. var. Control 0.62 0.60 1.22 0.7 2.0 1.3 texensis Non-watered 0.52 0.63 1.15 0.5 2.2 1.7 Average 0.57 0.62 1.18 0.6 2.1 1.5 C. chinensis Control 0.63 0.53 1.17 0.8 2.5 1.7 Non-watered 0.53 0.28 0.82 0.7 4.3 3.7

120 Average 0.58 0.41 0.99 0.8 3.4 2.7

C. Control 0.58 0.27 0.85 0.3 2.3 2.0 siliquastrum Non-watered 0.25 0.28 0.53 0.8 2.3 1.5 Average 0.42 0.28 0.69 0.6 2.3 1.8 Taxa 0.1360 0.0360 0.6441 0.6441 0.1367 0.4042 Treatment 0.0073 0.2747 0.0398 0.0398 0.6117 0.0635 Taxa x treatment 0.8197 0.4101 0.5880 0.5880 0.0347 0.2142

120

Plant dry weight during water deficit and recovery

Non-watered plants increased leaf dry weight less than control plants during the period of water deficit (Table 4.5). A significant interaction between taxa and treatment was observed for leaf dry weight increase during the recovery period. Non-watered plants increased more in leaf dry weight than control plants in all taxa, with the exception of

C. canadensis (OH) where the opposite was observed. Overall, total leaf dry weight

(combined water deficit and recovery period) was not affected by taxa or water deficit treatment.

Shoot dry weight increase during the period of water deficit was affected by taxa (Table

4.5); C. canadensis (OH) had the highest shoot growth, C. canadensis (OK) and C. canadensis var. texensis showed a slight reduction. During the recovery period, a significant taxa by treatment interaction occurred. Control C. canadensis plants from

(PA) and (OH) showed a relatively larger increase in shoot dry weight relative to non- watered plants. However, in all other taxa shoot dry weight increase was greater in previously non-watered plants. Total increase in shoot dry weight was affected by taxa.

Cercis canadensis (OH) had more than four times greater increase in shoot dry weight than C. occidentalis, C. canadensis (OK), C. canadensis var. texensis and C. chinensis.

Root dry weight was not affected by taxa or treatment during the period of water deficit(Table 4.6). During the recovery period, a significant interaction for taxa and treatment was found. Previously non-watered plants of C. canadensis (PA) and C. chinensis had greater root dry weight increase than control plants. Other taxa showed the opposite reaction, the increase in root dry weight during the recovery period was greater in control plants. Overall, control plants accumulated more root dry weight than plants

121

that went through the water deficit and recovery cycle. Total dry weight increase during the period of water deficit was significantly different among taxa (Table 4.6). The greatest difference was observed between C. canadensis (OH), which increased the most in dry weight, and C. canadensis var. texensis which increased the least. However, during the recovery period C. canadensis (PA), C. canadensis (OK) and C. canadensis var. texensis had the greatest increase in dry weight, while C. chinensis was unchanged. The total dry weight increase (water deficit and recovery period combined) was affected by taxa and water deficit treatment. Cercis canadensis (OH) increased most in dry weight over of the experiment while C. occidentalis, C. canadensis (OK), C. canadensis var. texensis and C. chinensis increased least. Overall, control plants increased more in dry weight than plants exposed to the water deficit and recovery cycle. There were differences in the shoot-root ratio during the period of water deficit due to differences among the taxa (Table 4.7). Cercis canadensis var. texensis increased in shoot-root ratio, due to a greater increase in shoot than root dry weight during the period of water deficit.

Cercis occidentalis produced more root than shoot dry weight during the period of water deficit as shown in a reduction in shoot-root ratio. During the recovery, period a significant treatment effect was observed for the shoot-root ratio. Both control and non- watered plants decreased in shoot-root ratio, however, non-watered plants decreased much less than control plants, indicating a greater shift toward shoot development during water deficit in non-watered plants. The change in leaf area during the water deficit was affected by both taxa and treatment (Table 4.7). Cercis canadensis (OH) increased most in leaf area. Sources of C. canadensis from Pennsylvania and Oklahoma, as well as C. canadensis var. texensis showed a reduction in leaf area. Control plants increased in leaf

122

area by 168 cm2, while non-watered plants reduced leaf area by 118 cm2. No significant differences between taxa or treatments were observed during the recovery period. During the experiment leaf area was significantly affected by taxa and treatment. The Ohio source of C. canadensis had the least reduction in leaf area while the source from

Pennsylvania lost more than six times as much as leaf area as C. canadensis (OH). Both control and non-watered plants lost leaf area, but non-watered plants showed a greater reduction than control plants.

123

Dry weight Leaves (g) Shoots (g) Taxa Treatment Water-deficit Recovery Total Water-deficit Recovery Total C. canadensis Control 3.1z -1.0 2.1 4.1 4.5 8.6 (PA) Non-watered -0.5 3.4 2.9 2.7 2.0 4.6 Average 1.3 1.2 2.5 3.4 aby 3.2 6.6 ab C. canadensis Control 3.5 3.0 6.5 3.3 6.6 9.9 (OH) Non-watered 3.8 -1.7 2.1 9.1 -2.6 6.4 Average 3.7 0.6 4.3 6.2 a 2.0 8.2 a C. Control 1.2 0.2 1.6 0.3 1.7 1.1

124 occidentalis Non-watered 0.2 2.8 3.1 -0.1 2.8 2.7

Average 0.7 1.5 2.3 0.1 b 2.2 1.9 c Continued

Table 4.5 Changes in leaf and shoot dry weight of seven Cercis taxa exposed to a water deficit and recovery cycle. Numbers represent means of six plants per treatment combination.

z Each value is the mean of six plant per treatment combination.

y Means within a column followed by different letters are significantly different from each other at α 0.0 using Tukey’s test of significance.

124

Table 4.5 continued

C. canadensis Control -0.3 1.6 0.8 0.1 0.9 0.9 (OK) Non-watered -2.4 2.4 -1.3 -2.0 3.5 1.6 Average -1.4 2.0 -0.3 -1.0 b 2.2 1.3 c C. canadensis Control 1.5 1.0 2.5 -0.1 0.7 0.6 var. texensis Non-watered -1.3 1.3 0.0 -0.7 3.6 2.9 Average 0.1 1.1 1.3 -0.4 b 2.1 1.7 c C. chinensis Control 5.5 -3.8 1.6 3.8 -2.5 1.2 Non-watered -0.3 2.4 1.4 -0.2 0.8 0.6

125 Average 2.6 -0.7 1.5 1.8 ab -0.9 0.9 c

C. Control 3.9 -2.2 1.7 1.5 0.9 2.4 siliquastrum Non-watered 2.4 1.7 4.1 0.5 2.4 2.9 Average 3.2 -0.2 2.9 1.0 b 1.6 2.6 bc Taxa 0.0563 0.659 0.1232 0.0003 0.2332 <0.0001 Treatment 0.0136 0.0121 0.5444 0.6028 0.9259 0.6646 Taxa x treatment 0.6899 0.0099 0.246 0.1154 0.0006 0.2578

125

Dry weight Roots Total Taxa Treatment Water deficit Recovery Total Water deficit Recovery Total C. canadensis Control 5.7z 0.3 6.0 10.0 6.9 16.9 (PA) Non-watered 3.5 3.0 6.5 1.0 12.3 13.3 Average 4.6 1.7 6.3 5.5 aby 9.6 a 15.1 ab C. canadensis Control 1.5 9.0 10.5 8.3 18.6 26.9 (OH) Non-watered 5.3 -0.1 5.1 18.1 -4.4 13.6 Average 3.4 4.5 7.8 13.2 a 7.1 ab 20.3 a C. Control 2.3 4.8 7.1 3.1 6.7 9.8

126 occidentalis Non-watered 4.6 1.9 6.5 4.8 7.5 12.3

Average 3.4 3.4 6.8 4.0 ab 7.1 ab 11.0 b Continued

Table 4.6 Changes in root and total dry weight of seven Cercis taxa exposed to a water deficit and recovery cycle. Numbers represent means of six plants per treatment combination.

z Each value is the mean of six plant per treatment combination.

y Means within a column followed by different letters are significantly different from each other at α 0.0 using the Waller- Duncan test of significance.

126

Table 4.6 continued

C. canadensis Control 3.7 5.4 9.1 6.0 7.8 13.9 (OK) Non-watered 3.5 2.3 5.9 1.2 10.5 5.8 Average 3.6 3.9 7.5 3.6 ab 9.2 a 9.8 b C. canadensis Control 1.9 8.9 10.7 1.2 10.0 11.2 var. texensis Non-watered 3.7 1.5 5.1 1.7 6.3 8.0 Average 2.8 5.2 7.9 1.5 b 8.2 a 9.6 b C. chinensis Control 4.4 0.5 4.9 13.1 -5.6 7.5 Non-watered 1.6 2.7 4.3 0.3 5.7 6.0

127 Average 3.0 1.6 4.6 6.7 ab 0.1 b 6.7 b

C. Control 2.5 4.0 6.4 7.9 2.6 10.5 siliquastrum Non-watered 4.7 2.0 6.7 7.6 6.4 14.0 Average 3.6 3.0 6.6 7.7 ab 4.5 ab 12.2 ab Taxa 0.8427 0.0760 0.1696 0.0377 0.0129 0.0002 Treatment 0.2157 0.0001 0.0051 0.3861 0.6413 0.0184 Taxa x treatment 0.0560 0.0004 0.1626 0.0602 <0.0001 0.0561

127

LAR, NAR and RGR

Water deficit had no effect on LAR, during the water deficit or the recovery period (data not shown). The NAR was not affected by taxa or treatment effect during the period of water deficit, but showed a treatment effect during the recovery period (Table 4.7). The

NAR of non-watered plants was (-0.141 mg cm-2 d-1) lower than control plants (0.003 mg cm-2 d-1) during the recovery period. Over the course of the experiment a significant taxa by treatment interaction was observed. In most taxa (C. canadensis (PA), C. occidentalis,

C. chinensis and C. siliquastrum) control plants had higher NAR than non-watered plants. However, the opposite was found for C. canadensis (OH) where the control plants had lower NAR than non-watered plants. In C. canadensis (OK) both control plants and non-watered plants had negative NAR values while control plants had lower NAR values than non-watered plants. Cercis canadensis var. texensis displayed negative NAR values for control plants, but positive values for non-watered plants. The RGR differed only for taxa during the period of water deficit (Table 4.7). Here, C. canadensis from Ohio had significantly higher RGR values than all other taxa.

128

NAR RGR mg/cm2/day mg/g/day Taxa Treatment Water deficit Recovery Total Water deficit Recovery Total C. canadensis Control -0.08z -0.13 4.32 7.7 72.1 79.8 (PA) Non-watered -1.49 -0.14 -1.63 -1.3 11.5 10.2 Average -0.78 b -0.14 1.35 3.2 b 41.8 45.0 C. canadensi. Control 0.69 -0.01 0.69 26.7 3.1 29.7 (OH) Non-watered 2.49 -0.24 2.25 24.7 -23.6 1.1 Average 1.59 a -0.12 1.47 25.7 a -10.3 15.4 C. Control -0.01 0.73 0.72 0.5 10.2 10.7

129 occidentalis Non-watered -0.17 -0.17 -0.34 8.0 9.4 17.3

Average -0.09 ab 0.28 0.19 4.2 b 9.8 14.0 Continued

Table 4.7 Net assimilation rate (NAR) and relative growth rate (RGR) of seven Cercis taxa exposed to a water deficit and recovery cycle.

z Each value is the mean of six plant per treatment combination.

129

Table 4.7 continued

C. canadensis Control -0.28 -0.09 -0.30 5.3 7.3 11.3 (OK) Non-watered -0.14 -0.11 -0.18 -2.4 8.4 3.9 Average -0.21 ab -0.10 -0.24 1.5 b 7.9 7.6 C. canadensis Control 0.00 -0.14 -0.13 0.3 11.1 11.4 var. texensis Non-watered 0.16 -0.02 0.14 3.8 4.4 8.1 Average 0.08 ab -0.08 0.00 2.0 b 7.7 9.8 C. chinensis Control 0.53 -0.02 0.51 12.0 -5.0 6.9 Non-watered 0.00 -0.02 -0.02 -0.7 6.2 4.4

130 Average 0.26 ab -0.02 0.24 5.6 b 0.6 5.7

C. Control 0.35 -0.03 0.32 12.1 2.9 15.0 siliquastrum Non-watered 0.22 -0.26 -0.04 6.6 11.0 17.6 Average 0.29 ab -0.14 0.14 9.4 b 7.0 16.3 Taxa 0.0877 0.4477 0.5137 <0.0001 0.5428 0.6225 Treatment 0.8382 0.0025 0.1051 0.3534 0.2048 0.1359 Taxa x treatment 0.4584 0.5312 0.0202 0.0750 0.6302 0.5961

130

Gas exchange

Net Photosynthesis (Pn) and stomatal conductance during the period of water deficit are presented relative to initial values. Cercis occidentalis and C. siliquastrum had the highest relative Pn at the beginning of the experiment while C. chinensis had the lowest

(Fig. 4.3 H-N). All taxa had lower relative Pn in non-watered plants than control plants.

At time of maximum water deficit the relative Pn of non-watered plants was reduced by

72% compared to 12% in control plants. After the recovery period, C. chinensis had the highest Pn, while C. siliquastrum had the lowest relative Pn. At the end of the recovery period, relative Pn was affected by treatment and taxa. In control plants Pn, was reduced to 78%, while non-watered plants were reduced to 59%. Cercis chinensis maintained the highest Pn rate while C. siliquastrum was reduced most. Overall, Pn was affected only by treatment. Control plants maintained a Pn rate of 83% of their initial value, while plants undergoing the water deficit andrecovery cycle maintained a Pn rate of 45%.

Stomatal conductance was affected by taxa and treatment at time of maximum water deficit. Cercis chinensis had the highest stomatal conductance (percent of the initial value, Fig 4.3 M) while C. occidentalis and C. canadensis (OK) decreased most (Fig. 4.3.

J and K). The non-watered treatment reduced plant stomatal conductance to 19% of the initial value while control plants maintained stomatal conductance at 64% of the initial value. After the recovery period, stomatal conductance was only affected by treatment.

Previously non-watered plants had a stomatal conductance reduced to 41% while control plants had a stomatal conductance of 66% of their initial value. Overall (water deficit and recovery period) control plants maintained a significantly higher stomatal conductance

(65% of their initial) value than non-watered and recovered plants (31%). 131

Chlorophyll fluorescence

All Cercis taxa had Fv/Fm values at or above 0.75 at the beginning of the experiment

(Fig. 4.3 H-N). However, significant differences among taxa were observed; C. occidentalis, C. chinensis and C. siliquastrum had the highest Fv/Fm values, while C. canadensis (OH) had the lowest Fv/Fm value. Only relatively small changes in Fv/Fm values were observed during the course of the experiment. At time of maximum water deficit, significant differences between treatments were found. Control plants had an

Fv/Fm value of 0.77 whereas non-watered plants had values of 0.73. Taxa affected by water deficit showed an incline in Fv/Fm values, however, non-watered plant still had significant lower Fv/Fm values (0.75) than control plants (0.77). There were also differences between the taxa; at the beginning of the experiment, C. siliquastrum and C. chinensis had the highest Fv/Fm values, while C. canadensis (OH) had the lowest.

132

Fig. 4.3 H. Cercis canadensis (PA)

25 0.5 0.9

0.4 20 0.8

0.3 15

0.2 0.7

Fv/Fm 10

Photosynthesis 0.1 Stomatal conductance Stomatal 0.6 5 0.0

0 0.5 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

Stomatal conductance control Photosynthesis control Chlorophyll fluorescence control

-2 -1 Fig. 4.3 H-N. Photosynthesis (μmol CO2 m leaf area s ), stomatal conductance (mol -2 -1 H2O m leaf area s ) and chlorophyll fluorescence of seven Cercis taxa exposed to a water deficit and recovery cycle. Solid lines represent the control treatment; dashed lines represent the non-watered treatment. Arrows represent time of re-watering. Photosynthesis and stomatal conductance values represents one leaf from six single plant replications, chlorophyll fluorescence values represent the average of three leaves per single plant replication.

Continued 133

Fig.4.3 condinued: I. Cercis canadensis (OH)

25 0.5 0.9

0.4 20 0.8

0.3 15

0.2 0.7

Fv/Fm 10

Photosynthesis 0.1 Stomatal conductance Stomatal 0.6 5 0.0

0 0.5 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

Stomatal conductance control Photosynthesis control Chlorophyll fluorescence control

Fig.4.3 J. Cercis occidentalis

25 0.5 0.9

20 0.4 0.8

15 0.3

0.7

Fv/Fm 10 0.2

Photosynthesis

Stomatal conductance Stomatal 0.6 5 0.1

0 0.0 0.5 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

Stomatal conductance control Photosynthesis control Chlorophyll fluorescence control

Continued 134

Fig. 4.3 continued: K. Cercis canadensis (OK)

25 0.5 0.9

20 0.4 0.8

15 0.3

0.7

Fv/Fm 10 0.2

Photosynthesis

Stomatal conductance Stomatal 0.6 5 0.1

0 0.0 0.5 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

Stomatal conductance control Photosynthesis control Chlorophyll fluorescence control

Fig. 4.3 L. Cercis canadensis var. texensis

25 0.5 0.9

20 0.4 0.8

15 0.3

0.7

Fv/Fm 10 0.2

Photosynthesis

Stomatal conductance Stomatal 0.6 5 0.1

0 0.0 0.5 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

Stomatal conductance control Photosynthesis control Chlorophyll fluorescence control

Continued 135

Fig. 4.3 continued: M. Cercis chinensis

25 0.5 0.9

20 0.4 0.8

15 0.3

0.7

Fv/Fm 10 0.2

Photosynthesis

Stomatal conductance Stomatal 0.6 5 0.1

0 0.0 0.5 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

Stomatal conductance control Photosynthesis control Chlorophyll fluorescence control

Fig. 4.3 N. Cercis siliquastrum

25 0.5 0.9

20 0.4 0.8

15 0.3

0.7

Fv/Fm 10 0.2

Photosynthesis

Stomatal conductance Stomatal 0.6 5 0.1

0 0.0 0.5 7/27/09 8/3/09 8/10/09 8/17/09 8/24/09 Date

Stomatal conductance control Photosynthesis control Chlorophyll fluorescence control

136

Observations

At maximum water deficit signs of water stress varied with taxa. Most plants of C. canadensis (PA) had abscised the main shoot terminal and showed leaf blades which were folded inwards along the main vein (Fig. 4.3 N). Petioles remained upright but the leaf blade fell to a vertical position with the hinge point at the upper pulvinus at the base of the leaf lamina. Older leaves were displayed more vertically than younger leaves.

Cercis canadensis (OH) showed wilted and senesced main shoot terminals but did not usually abscise these (Fig. 4.3 O). Leaf blades appeared wilted and were in a vertical position with the leaf petioles pointing downwards. The main shoot apexes of C. occidentalis showed no signs of drought-stress; but could then suddenly senescence or abscise (Fig. 4.3 P). Overall the leaves turned lighter green and showed a slight inward rolling of the leaf edge. At maximum water deficit all leaves were in a vertical position, however, petioles on the lower part of the main shoot axis were positioned downward, while leaves up higher on the shoot axis remained upright. Cercis canadensis (OK) displayed wilted terminals, at maximum water deficit, but did not usually senesce or abscise (Fig. 4.3 Q). The leaves, especially on the upper part of the shoot axis were rolled inwards and although the petioles remained upright, the leaves were positioned vertically.

The main shoot terminals of C. canadensis var. texensis showed no signs of water stress

(Fig. R). The leaves themselves did not appear wilted, but were positioned vertically with petioles in a downward position. Cercis chinensis responded similar to water deficit as C. canadensis var. texensis in that the terminal and the leaf blades did not show signs of water stress (Fig. 4.3 S). However, opposite to C. canadensis var. texensis leaf response, the petioles in C. chinensis were positioned downwards. Leaves in the lower part of the

137

main shoot axis were positioned more vertically than on the upper part of the axis. Cercis siliquastrum showed wilted terminals, which typically senesced and abscised early during water deficit (Fig. 4.3 T). Leaves, especially on the upper part of the main shoot axis, rolled inwards as water deficit increased. However, most leaves showed no signs of wilting for a long period of time. When senescence occurred, it progressed rapidly overnight and mostly as marginal leaf scorching or as necrotic patches on otherwise green leaves.

138

Fig. 4.4 O-U

O. Cercis canadensis (PA)

P. Cercis canadensis (OH)

Fig. 4.4 Morphological signs of water deficit stress of seven Cercis taxa. Shown are responses of the main shoot terminal, the leaf blade and the leaf position on the main shoot axis.

Continued

139

Fig. 4.4 O-U, continued

Q. Cercis occidentalis

R. Cercis canadensis (OK)

S. Cercis canadensis var. texensis

Continued 140

Fig. 4.4 O-U. continued

T. Cercis chinensis

U. Cercis siliquastrum

141

4.5 Discussion

Non-watered plants at the peak water-deficit harvest experienced lower soil water potentials as indicated by a soil moisture potential of -8.55 to -185.28 MPa than watered plants.

Water use

Water use (g) decreased during the period of water deficit in non-watered plants compared to the initial water use but increased in watered control plants. At the end of the recovery, however, both treatments used more water than at the beginning of the experiment. Control plants used significantly more water than non-watered plants throughout the whole experiment. However, the higher water use of both treatments at the end of the experiment is likely due to greater plants size. Control plants increased more in total plant dry weight during the experiment than non-watered plants, indicating that reduced soil moisture reduced plant growth.

Gas exchange

Plants under water deficit conditions usually show a related decrease between stomatal conductance and photosynthesis, since the stomata control photosynthesis through both regulation of transpiration and absorption of carbon dioxide (Ni and Pallardy 1992), which contributes to the reduced growth rate under water deficit conditions. Mesic- adapted species typically exhibit greater and more rapid decrease in photosynthesis and stomatal conductance rates than xeric-adapted species (Bahari, Pallardy and Parker 1985,

Abrams, Schultz and Kleiner 1990). The relatively higher rates of photosynthesis in xeric

142

species, even under drought conditions, have often been attributed to the maintenance of higher stomatal conductance through means of elastic or osmotic adjustment, deep rooting ability or leaf structural characteristics (Bahari et al. 1985, Abrams et al. 1990).

Root length varied widely within the Cercis taxa in this study (Chapter 2). Cercis occidentalis, considered adapted to a xeric environment, had the lowest root length; while

C. chinensis (considered mesic adapted) had a root system more as twice as long. Cercis occidentalis and C. griffithii, both considered xeric adapted species, had the most efficient root system in terms of water use per day per cm2 leaf area, while C. chinensis as mesic adapted species had the least efficient root system. However, no consistent patterns regarding the water use efficiency could be found among the other taxa.

In our experiment all taxa showed a decrease in stomatal conductance and net photosynthesis with the onset of water deficit. This response to limited water availability is typical for drought avoiding ‘water savers’. Plants minimize water loss, to maintain high leaf water potentials as long as possible to prevent tissue damage. Drought tolerance was also expressed; stomatal conductance and net photosynthesis rapidly returned to near pre-water deficit levels after seedlings under water deficit treatment were re-watered.

Supplementing the drought tolerance mechanism was paraheliotropism, which reduced light interception and helped to preserve the photosynthetic apparatus. Unique characteristics of paraheliotropism were observed among the taxa.

Two xeric-adapted species from, C. occidentalis (CA) and C. siliquastrum (Italy) showed much higher initial photosynthesis rates and also maintained higher photosynthesis rates during the period of limited water availability. This is typical for drought resistant plants,

143

which often have higher gas exchange rates compared to drought sensitive plants (Adams et al. 1990). According to Krueger and van Rensburg (1995) survival and recovery after a drought are directly related to the maintenance of positive photosynthesis during a slowly developing drought. Opposite to the other taxa, non-watered C. occidentalis and C. siliquastrum showed a general decline in photosynthesis and stomatal conductance toward the end of the experiment, as did the watered control. While non-watered plants recovered to levels of control plants at the end of the experiment, pre-water deficit values were not reached. This may be due to seasonal leaf aging. This adaptation comes at the cost of the early succession of growth. For the other taxa, photosynthesis decreases rapidly in response to water deficit and is an apparent adaptation to an environment where rain may occur sporadically over the growing season. Cercis occidentalis

(California) and C. siliquastrum (Italy) are to be allopatric species and show convergent evolution. Both species are native to Mediterranean climates and employ similar life strategies. However, their geographic separation indicates distinct evolutionary histories.

Additional convergent characteristics are plant growth and leaf morphological characteristics (Chapter 2) such as a short shruby growth habit and small, ovate-round leaves.

Chlorophyll fluorescence

Chlorophyll fluorescence measurements have been used to evaluate the integrity of photosystem II during water deficit and after recovery. A decrease in the Fv/Fm value under stress have been attributed to the inactivity of the PSII reaction centers (Hao et. al

1999). At maximum water deficit, significant differences between treatments were

144

observed when averaged over taxa. However, while C. canadensis (OH), C. canadensis

(OK) and C. chinensis showed a great decrease in Fv/Fm values, other taxa did not. In taxa that did not alter the PSII function (C. canadensis (PA), C. occidentalis and C. siliquastrum), the PSII system can be considered highly drought resistant.

However, after re-watering chlorophyll fluorescence (Fv/Fm) reached 95% of the initial values, showing that a near complete reactivation of the photosynthetic processes occurred. These results showed that the photosynthetic apparatus of Cercis taxa was very resistant to an 8 d water stress treatment; either through a highly drought resistant PSII system or through a rapid recovery once the stress has ceased. Although the water deficit treatment was short in duration, it was severe, reaching a soil moisture potential of at least -0.87 MPa. We conclude that one common drought resistance mechanism of these

Cercis taxa was the ability of plants to withstand drought stress without altering the function of the PSII system and/or the capacity of plants to quickly restore the damage to the photochemical apparatus.

Observations

High irradiance can lead to photoinhibition, especially under drought stress conditions when stomata are closed (Bjoerkman 1981, Powles 1984). Therefore, mechanisms which reduce the incident irradiance can protect the photosynthetic system from damage caused by excess excitation energy from the light reactions (Ludlow and Bjoerkman 1983).

One mechanism for protection of the photosynthetic system is paraheliotropism, the avoidance of direct exposure to sun. Paraheliotropism is common in legumes such as beans and is also known in Cercis (Reed 1987). The minimization of light interception 145

through the vertical orientation of the leaf blade minimizes light interception (Yu and

Berg 1994), and leaf temperature (Ehleringer and Forseth 1980) and transpirational water loss (Gamon and Pearcy 1989) compared to horizontally displayed leaves. Ludlow and

Bjoerkman (1983) found that in drought-stressed Macroptilium atropurpureum, a legume native to Mexico, paraheliotropic leaf movement protected the plants from high temperature damage, photoinhibition and the interactive effects of high temperature and excess light. Paraheliotropic leaf movement has also been suggested as a factor for increasing water-use efficiency (Ehleringer and Forseth 1980, Forseth and Ehleringer

1983, Raeini-Sarjax et al. 1997). Bielenberg et al. (2003) demonstrated on Phaseolus that paraheliotropism also acts through shifting the water-use efficiency towards more favorable values, allowing the plant to postpone the growth- or survival-limiting effects of drought. However, due to the pure observational nature of the data, and non-significant stress-treatment effect during the period of water deficit, an effect of paraheliotropism on

WUE was not established.

The petiole of Cercis leaves consists of a lower pulvinus at the base, a petiole proper, and an upper pulvinus at the base of the leaf lamina (Owens 1998). The movement of pulvini is promoted by environmental factors including high temperatures, high light and low water potential (Yu and Berg 1994). Darwin (1881) observed paraheliotropism in many arid environments species. It was found that species and even varieties vary in their leaf movement (Wofford and Allen 1982, Travis and Reed 1983). Therefore, even closely related taxa may vary in their paraheliotropic response depending on which response would be beneficial in their environment (Yu and Berg 1994). Paraheliotropic leaf movements respond rapidly to environmental stresses and are completely reversible,

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unlike developmentally controlled responses (Yu and Berg 1994). In combination with other characteristics, such as small leaves and leaf area, a high root to shoot ratio and the ability to tolerate high temperature, paraheliotropism increases a plants drought resistance. The Cercis taxa observed here, showed varying degrees of paraheliotropism in combination with other protective mechanism as wilting or rolling of the leaf.

Paraheliotropism is mostly observed in arid environments, and little is known about it outside of these environments. The display of paraheliotropism in mesic areas, especially in C. canadensis in the north-eastern U.S. in understory conditions, may be explained by the molecular taxonomy of Cercis. A study by Davis et al. (2002) suggested that North

American Cercis share a common xerophytic ancestor and that the north-eastern redbud represents a mesophytic reversal to the conditions present in the ancestor of the genus.

The eastern redbud may have retained certain xerophytic traits despite its typical understory habitat.

I conclude that all of the taxa in our study are drought-resistant and employ several common drought resistant mechanisms. One is the ability of plants to withstand drought stress without altering the function of the PSII system and/or the capacity of plants to quickly restore the damage to the photochemical apparatus. Another one is paraheliotropism, which allows the plants to minimize light exposure and leaf temperature. All of the taxa studied are drought-resistant; however, in Chapters 2 and 3 experiments, the taxa displayed unique morphological and growth characteristics. In this study, conducted with older and larger plants, the taxa tended to respond similarly to increasing water deficit.

147

The most commercially produced redbud in the United States is the eastern redbuds (C. canadensis). This study showed that other fast-growing taxa, like C. canadensis var. texensis or C. chinensis, could be timely produced, while on the other hand offering the advantage of increased drought resistance. Cercis occidentalis and C. siliquastrum, are very drought resistant due to their ability to maintain higher rates of Pn and stomatal conductance than other taxa. However, they appear to succeed growth early due to seasonal leaf aging.

There is no evidence for clinal variation among the North American taxa. This may be due to insufficient sampling of provenances.

Even though only a limited number of plants were used for this experiment; the Cercis taxa displayed overall a large within species variation. Especially C. canadensis var. texensis were variations in leaf surface (glabrous or glossy) and differences in plant growth habit were observed. Cercis canadensis var. texensis would be an excellent candidate for inducing drought resistance into a breeding program based on its drought resistance and its growth habit which is more tree like than other xeric taxa in this study.

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Chapter 5

DISCUSSION

149

Three experiments were conducted; one study with older (Chapter 2), but still relatively young plant under non-moisture conditions to describe the taxa used. A second experiment (Chapter 3) was conducted with juvenile plants under non-limining soil moisture conditions to explore above-and-below ground biomass allocation and water use. A third experiment (Chapter 4) was conducted under limiting soil moisture conditions with older but still relatively young to study the effect of water deficit on plant morphology and physiology.

Morphological and physiological evidence was obtained that showed that the taxa studied are different but it can not be determined if they are separate species as there is no knowledge of reproductive barriers and the degree of gene flow among the taxa.

All Cercis taxa were drought resistant and displayed both drought avoidance and drought tolerance mechanisms. Unique morphological and physiological characteristics were seen under non-limiting soil moisture conditons. However, taxa responded similarly under limiting soil moisture conditions. I found little evidence for clinal variation among the

North American taxa, possibly due to limited number of seed sources studied.

Based on the results of these three experiments, there is no common suite of characteristics that would explain adaption to mesic or xeric environments. All the taxa studied were drought resistant and used a unique set of characteristics of both avoidance and tolerance mechanisms to resist drought.

Water is one of the principle factors determining the species composition of a given site.

Drought is one of the most encountered stresses by plants and the most injuries one due to its effects on nearly every physiological process (Kramer 1987, Cregg 2004). In urban 150

and suburban environments drought stress is magnified. However, trees are often selected for their aesthetic contribution to the landscape, with little consideration of how they can perform in a stressful urban environment. The variety of plant characteristics contributing to drought resistance as well as the variation in planting site characteristics makes plant selection for urban sites difficult. Although irrigation can mitigate the impact of drought stress, increasing water prices, limited availability of ground water, ground water pollution, and water restrictions and its politics, it is becoming less of an option (Schuch and Burger 1987, Knox 1989). Selecting trees with improved drought tolerance and reduced water use may be the best strategy to improve survival, growth and health of trees in urban and suburban landscapes (Cregg 2004).

The aim of this study was to investigate the inter-taxa variation in drought resistance and water use efficiency existing within the genus Cercis, thereby providing information as to their usefulness for plantings in urban landscapes and to facilitate breeding for improved water use and drought resistance. A better understanding of the morphology and physiology and their adaptability to drought stress will allow for more appropriate selection of taxa for specific sites (Ranney et al. 1990).

The genus Cercis includes 10 recognized species; however, the taxonomy of Cercis has undergone several changes in concept and the exact number of species remains controversial.

In the first study growth habits and leaf characteristics of seven Cercis taxa (two additional taxa for leaf characteristics) were measured and analyzed using principal component analysis. Cercis taxa described here showed characteristics typical for the

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taxa/species as described in the literature. However, often times the literature is inconclusive on certain characteristics of some taxa. Great variation in plant growth and leaf characteristics was observed among Cercis taxa and could be used to separate taxa.

A wide variety of adaptations to local environments were found in the taxa described in this study. Differences in branching habit, leaf size and shape as well as differences in leaf surface were observed. However, under the conditions of these experiments with juvenile plants and under non-limiting soil moisture conditions, clinal variation does not appear to exist among the North American taxa.

Cercis taxa could be divided into three groups based on plant growth habit. Cercis occidentalis, C. siliquastrum and C. griffithii were multi-stemmed and could be grouped based on their medium height and branch number. Cercis canadensis (OK), Cercis canadensis var. texensis and C. chinensis, grew tall with branching starting above 40 % of the height of the main shoot axis. Cercis canadensis var. mexicana was visibly different from other taxa, mainly due to its relatively low height and its densely branched habit. Plant branch architecture likely influences stem flow; a process through which the ground area around the plants stem receives additional moisture, while its shade limits competition (Steinbuck 2002) and could be considered as a xeric-site adaptation. The increased vegetation cover of highly branched taxa should allow them to collect more water through stem-flow, then, in contrast, C. chinensis; a mesic-adapted species which grows strictly upright, had very few first order branches and usually no branches of higher order.

Cercis occidentalis, C. siliquastrum and C. canadensis var. mexicana had similar leaf morphology; relatively small in size, with a rounded leaf apex, resulting in a common 152

grouping on via PC analysis. Other taxa displayed mostly acuminate to acute leaf apices.

These results support the finding of Donselman and Flint (1982) and Fritsch et al. (2009) that plants from southern origin had smaller leaves and a more rounded leaf apex than those from northern origin. Larger leaves would increase the interception of solar radiation and therefore be an advantage under low light conditions (under story). In dry areas, under high light conditions, however, maximizing light interception has no adaptive advantage and may have presented a selection pressure for smaller leaves

(Donselman and Flint 1982, Fritsch et al. 2009).

Cercis occidentalis and C. siliquastrum clustered together in the principal component analysis, even they are allopatric species. One of the main common characteristic of

Cercis occidentalis and C. siliquastrum, is the glaucousness of their leaf surfaces.

Genetic differences in cuticular effectiveness were found and Nagarajah (1979) suggested that cuticular effectiveness could be improved through selection and breeding. However, these highly reflective properties are a constitutive trait, and while beneficial during water deficit, at times of non-limited soil moisture these properties often reduce photosynthesis and ultimately growth (Sanchez et al. 2001). Both species are native to xeric environments with high light and temperature conditions, where glaucousness can decrease cuticular water loss and increase light reflection. Cercis siliquastrum is native to the Mediterranean where dry summers and rainy winter prevail. However, this

Mediterranean climate does not only occur in the Mediterranean region but also in the native range of C. occidentalis (California). This development of similar characteristics in geographicly separated regions suggests convergent evolution.

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Some taxa in this experiment displayed the characteristics typically associated with their adaptation to mesic or xeric environments. However, taxa often displayed a combination of characteristics typically associated with mexic and xeric environments.

The second experiment explored above-and-below ground biomass allocation and water use. The principal component analysis suggests clinal variation among the North

American taxa, C. canadensis (OH), C. occidentalis, C. canadensis (OK) and C. canadensis var. texensis, based on loadings on PC1 (plant growth and water use per unit plant mass/area). No clustering was evident with regard to the other taxa under the conditions of this experiment (juvenile plants and non-limiting soil moisture conditions).

Root length, is constitutive trait influencing drought resistance. Maximum root depth depends on root length and the interaction of the plant with its environment. A deep and wide-spreading root system allows the plant to maximize water uptake from the soil when grown in a common, non soil-moisture limiting conditions. Similar aged Cercis taxa in this study varied widely in root length. Cercis occidentalis, considered adapted to a xeric environment, had the lowest root length; while C. chinensis (considered mesic adapted) had a root system more as twice as long. Cercis occidentalis also had the lowest specific root length (SRL, g root dry weight per unit root length). Low SRL results from shorter root length (smaller root surface area) per unit carbon invested. Some studies have found that fast-growing species have a higher SRL than species from more xeric environments (Ryser and Eek 2000; Craine et al. 2001, Nicotra et al 2002; Comas and

Eissenstat 2004), while other studies have shown that species from xeric environments have higher SRL than species from mesic environments (Poot and Lambers 2003,

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Tjoelker et al. 2005). In this study there was no difference in SRL among the taxa, however, C. occidentalis had the lowest SRL, while C. siliquastrum, another xeric adapted species had the highest (twice as high as C. occidentalis). The lack of significant difference in SRL may be due to a small sample size. Different life strategies become visible when plant size and water use were compared among taxa. Cercis chinensis and

C. canadensis var. texensis were the tallest plant, had the greatest leaf area and total plant dry weight. The seedlings of both taxa used high amount of water per day. However, C. siliquastrum, which is relatively smaller in height and has less leaf area, but comparable total plant dry weight had the highest water use per seedling per day of all taxa. The tall taxa, have high above-to-below ground biomass ratios (shoot-root ratio, ‘root length-to- leaf area ratio’,‘root surface-to-leaf area ratio’). Cercis siliquastrum was able to meet its water demand because of its much smaller size, significantly greater root surface and

‘root length-to-leaf area ratio’. However, no consistent pattern of plant growth or water use characteristics employed by mesic- or xeric-adapted taxa was found. Each taxon expressed a unique set of characteristics.

The objective of the third study was to investigate water use, gas exchange, chlorophyll fluorescence as well as plant growth in seedlings of Cercis taxa from different origins when submitted to a water deficit and recovery cycle and to determine the drought resistance mechanisms and the relative drought resistance among the taxa. Drought as a multi-dimensional stress, affects virtually all physiological processes directly or indirectly (Kramer 1987, Cregg 2004). Stomatal closure is among the earliest plant responses to water deficit and the most important factor in controlling carbon fixation

(Yordonov et al. 2000). A reduction in photosynthesis may be due to stomatal limitations 155

and/or to damage to the reaction centers of photosynthesis I and II (inhibition of the photosynthetic apparatus (Yordanov et al. 2000, Pallardy 2008)). In drought stressed- plants changes in the photochemical efficiency can be assessed by analysis of chlorophyll a fluorescence associated with PSII.

Mesic-adapted species typically exhibit greater and more rapid decrease in photosynthesis and stomatal conductance rates under drought stress than xeric-adapted species (Bahari, Pallardy and Parker 1985, Abrams, Schultz and Kleiner 1990). In our experiment all taxa showed a decrease in stomatal conductance and net photosynthesis with the onset of drought. This response to drought is typical for drought avoiding ‘water savers’. Plants minimize water loss, to maintain high leaf water potentials as long as possible to prevent tissue damage. However, plants also expressed drought tolerance mechanisms, since stomatal conductance and net photosynthesis rapidly returned near pre-water deficit levels once plants were re-watered.

Cercis occidentalis and C. siliquastrum, both considered xeric-adapted species, showed much higher initial rates of photosynthesis and stomatal conductance under water deficit.

As in other taxa, photosynthesis and stomatal conductance decreased immediately upon exposure to drought; however, relatively higher rates were maintained. These relatively higher rates of photosynthesis have been reported in the literature as typical for xeric- adapted species (Bahari, Pallardy and Parker 1985, Abrams, Schultz and Kleiner 1990).

Cercis occidentalis and C. siliquastrum are native to regions with Mediterranean climates and employ similar life strategies. The species displayed great similarities in morphological and physiological characteristics and in their response to water deficit.

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Cercis occidentalis and C. siliquastrum are allopatric species; and represent and example of convergent evolution. However, according to Davis et al. (2002) both species have a common ancestor. The vicariance of a xerophytic ancestor ranging over eastern North

America, western North America and western Eurasia resulted in C. occidentalis in western America, and a xerophytic ancestor of C. canadensis and C. siliquastrum ranging over western Eurasia and eastern North America. The vicariance of this xerophytic ancestor resulted in C. canadensis in eastern North America and C. siliquastrum in western Eurasia.

Chlorophyll fluorescence measurements have been used to evaluate the integrity of photosystem II under water deficit conditions and after recovery. Decreases in the Fv/Fm value under stress have been attributed to the inactivity of the PSII reaction centers (Hao et. al 1999). At maximum water deficit, significant differences between treatments were observed. Cercis canadensis (OH), C. canadensis (OK) and C. chinensis showed a great decrease in Fv/Fm values, but also recovered until the end of experiment to values of at least 92 % of the initial values. Taxa that did not alter the PSII function (C. canadensis

(PA), C. occidentalis and C. siliquastrum), can be considered highly drought resistant.

I conclude that all of the taxa in our study are drought-resistant and employ several common drought resistant mechanisms. One common mechanism was the ability of plants to withstand drought stress without altering the function of the PSII system and/or the capacity of plants to quickly restore the damage to the photochemical apparatus.

157

Another mechanism for protection of the photosynthetic system is paraheliotropism, the avoidance of direct exposure to sun. Paraheliotropism is common in legumes such as beans and is also known in Cercis (Reed 1987). The Cercis taxa observed here, showed varying degrees of paraheliotropism in combination with other protective mechanism as wilting or rolling of the leaf. Paraheliotropism is mostly observed in arid environments, and little is known about it outside of these environments. The display of paraheliotropism in mesic areas, especially in C. canadensis in the north-eastern U.S. in understory conditions, may be explained by the molecular taxonomy of Cercis. A study by Davis et al. (2002) suggested that North American Cercis share a common xerophytic ancestor and that the north-eastern redbud represents a mesophytic reversal to the conditions present in the ancestor of the genus. The eastern redbud may have retained certain xerophytic traits despite its typical understory habitat.

The most commercially produced redbud in the United States is the eastern redbuds (C. canadensis). This study showed that other fast-growing taxa, such as C. canadensis var. texensis or C. chinensis, could be timely produced, while on the other hand offering the advantage of increased drought resistance. Cercis occidentalis and C. siliquastrum, are very drought resistant due to their ability to maintain higher rates of Pn and stomatal conductance than other taxa. However, they appear to succeed growth early due to seasonal leaf aging.

The persistence of the leaf characteristics under non-limiting soil moisture conditions as shown in indicates thar the characteristics typical of the taxa grown in their native environment are under genetic control. The fact that some characteristics on the Eastern

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North American taxa espressed great variability indicates introgression of these charateristics and that the taxa are still in the process of speciation.

The genus Cercis appears to be a good candidate for genetic improvement for use in stressful urban sites. C. canadensis var. texensis would be an excellent candidate for inducing drought resistance into a breeding program. Variations were observed in leaf surface (glabrous or glossy) and plant growth habit. Cercis canadensis var. texensis showed no damage of the photosynthetic system during the drought period and can be considered highly drought resistant. It also has a more tree like growth habit and faster growth rate than other xeric taxa in this study.

Not addressed in this study was seedling mortality, which is a significant factor in seedling survival in a natural environment. Thuse, there may have been individuals studied which may not be represented in native populations. However, the seedlings likely represent the range of genetic diversity withing the particular seed sources studied.

Also, the water deficit experiment was short term and may not representative of water stress conditions of native environments.

Finally, the experiments did not measure the amount of morphological plasticity within the taxa. However, the Chapter 4 experiment did give insight into physiological plasticity.

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